A metabolomics approach for the evaluation of Ficus benghalensis female in vivo reproductive effects relative to its metabolite fingerprint as determined via UPLC-MS and GC-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Ficus benghalensis, commonly known as Banyan Fig, is the national tree of India and its aerial roots are used traditionally to treat female reproductive disorders. However, despite this traditional use, no pharmacological evidence could be traced supporting this use. Additionally, no comprehensive metabolite profiling was reported for F. benghalensis aerial roots. AIM OF THE STUDY: This study attempts to justify biochemically the traditional use of F. benghalensis aerial roots in treatment of female reproductive disorders and in relation to its secondary metabolite profile. MATERIALS AND METHODS: Total ethanol extract (TEE) and subfractions [petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and n-butanol (BUF] were prepared from air-dried powdered aerial roots of F. benghalensis. Detailed in-vivo investigation of the hormonal activity and action mechanism of the total ethanol extract and subfractions was carried out through evaluation of estrogenic and gonadotropic activities. The estrogenic activity was evaluated on ovariectomized immature female rats through estimating uterine weight, vaginal cornification and serum estradiol level along with histological examination of uteri. The gonadotropic activity was measured by assay of follicle stimulating hormone (FSH) and luteinizing hormone (LH) like activities. Total follicular and corpora lutea counts in immature female rats were used to determine FSH and LH like activities, respectively in addition to histological picture of the genitalia. Comprehensive non-targeted metabolite profiling was carried out for the TEE and subfractions using UPLC-HRMS in negative and positive ionization modes. UPLC-MS fingerprint was subjected to principal component analysis (PCA) and partial least squares analyses to correlate the bioactivities to specific chemical constituents in F. benghalensis different subfractions. GC-MS was further used for non-polar silylated fractions. RESULTS: Results revealed that only the non-polar PEF and CHF displayed moderate estrogenic and FSH-like activities but with no LH-like activity. Metabolites profiling via (UPLC-HRMS) and multivariate PCA analysis enabled identification and comparison of various chemical classes in F. benghalensis extract and fractions. The active non-polar fractions revealed nearly similar metabolites profile being composed of isoflavonoids, triterpenes, sterols, fatty acids and cyclic peptides. In contrast, polar fractions were more abundant in apocarotenoids, fatty acyl amides, hydroxybenzoates and hydroxycinnamates in addition to two lignans. PLS analysis revealed strong correlation between hydroxylated fatty acids and pyranoisoflavones with estrogenic and FSH-like activities. GC-MS analysis was further employed for non-polar fractions profiling revealing for their enrichment in fatty acids/esters, terpenes, organic acids and phenolics. CONCLUSION: This is the first study to rationalize the use of F. benghalensis aerial root traditionally in treatment of gynecological disorders, revealing that the petroleum ether and chloroform non-polar subfractions of F. benghalensis showed estrogenic and FSH-like activity with absence of LH-like activity. This biological activity could possibly be attributed to its metabolites profile of isoflavonoids, fatty acids, triterpenes, sterols and cyclic peptides identified via UPLC-MS and GC-MS techniques. Consequently, F. benghalensis aerial roots should be used with caution in traditional treatment of female infertility or other reproductive disorders.

Phytochemical characterization and hepatoprotective effect of active fragment from Adhatoda vasica Nees. against tert-butyl hydroperoxide induced oxidative impairment via activating AMPK/p62/Nrf2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Adhatoda vasica Nees., which existed in a large; number of Tibetan medicine prescriptions for hepatopathy, used as an adjuvant to treat liver diseases. HYPOTHESIS/PURPOSE: Oxidative stress is the key player in the development and progression of liver pathogenesis. In recent years, research is increasingly being focused on exploitation of the active components from medicinal plants to combat the liver oxidative injury. In our study, we aimed to screen the active principles from A. vasica and clarify whether they could relieve oxidative damage induced by tert-Butyl hydroperoxide (t-BHP) and its potential mechanism via activating AMPK/p62/Nrf2 pathway. MATERIALS AND METHODS: Ultra performance liquid chromatography (UPLC) was adopted for analysis of chemical composition in the extracts. Furthermore, the antioxidant activity of the fractions was evaluated using DPPH, ABTS and reducing power assay. Along with this, the compounds in this fraction with highest antioxidant activity were analyzed using UPLC-MS. Based on this, the condition for extracting flavonoids of this subfraction was optimized via response surface method. CCK-8 assay was used to detect cell viability. Detection kits were used to measure the activity changes of AST, ALT, LDH and CAT as well as MDA and GSH levels induced by t-BHP. Detection of reactive oxygen species (ROS) production was used DCFH-DA probe. DAPI staining and flow cytometry was used to detect cell apoptosis. In terms of the mechanistic studies, the expression of proteins involved in AMPK/p62/Nrf2 pathway was measured using western blotting. RESULTS: Eventually, 70% ethanol extract from leaf of A. vasica was chosen due to its highest active components compared with other extracts. Further, ethyl acetate fraction derived from 70% ethanol extract in A. vasica (AVEA) possess highest ability for scavenging DPPH and ABTS free radicals as well as strongest reducing power than other fractions. Chemical composition analysis showed that AVEA contained 17 compounds, including 1 quinazoline alkaloid, 12 flavonoid-C-glycosides and 4 flavonoid-O-glycosides. In addition, the conditions (ratio of solid-liquid 1:14, the concentration of ethanol 73%, and the temperature 65 °C) were selected to enrich the flavonoids in AVEA. Furthermore, AVEA could attenuate t-BHP induced hepatocyte damage via increasing the cell viability, restoring abnormal the activities of AST, ALT, LDH and CAT as well as the levels of MDA and GSH. ROS fluorescence intensity was reduced by AVEA. Meanwhile, it could inhibit the cell apoptosis of BRL 3 A cells, as evidenced by restoration of cell morphology and decreasing the number of apoptotic cells. Further mechanistic studies indicated AVEA could promote p-AMPK expression to further induce autophagy adaptor-p62 protein expression, which could autophagic degradation of Keap1, leading to Nrf2 release and translocation into nucleus to induce antioxidant genes (HO-1, NQO-1, GCLC and GCLM) expression. CONCLUSION: In our study, AVEA was first to screen as the active fraction in A. vasica with alkaloids and abundant flavones. Moreover, the fraction potentiates its beneficial aspect by displaying the protective role on relieving t-BHP induced oxidative stress and activating AMPK/p62/Nrf2 pathway. AVEA helps maintain the redox homeostasis of hepatic cells and could be considered as an effective candidate against oxidative stress related liver disorders.

Phenols fragment of Veronica ciliata Fisch. ameliorate free radical-induced nonalcoholic fatty liver disease by mediating PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Veronica ciliata Fisch. is used in numerous of Tibetan medicine prescriptions because of its hepatoprotective effect. AIMS OF THIS STUDY: Here, we aimed to investigate the hepatoprotective effect and mechanism of phenolic fraction (PF) of V. ciliata Fisch. on liver injury induced by free radical. MATERIALS AND METHODS: BRL 3A cells were pre-treated with PF and luteolin (Lut) following tert-butyl hydroperoxide (t-BHP) treatment. The cell viability, lactate dehydrogenase (LDH) levels, reactive oxygen species (ROS) generation, apoptosis, cell cycle and autophagy were analyzed. Apoptotic, inflammatory, and autophagy,- related proteins were analyzed using Western blotting. The combination of molecular docking and drug affinity targeting experiments (DARTS) were first utilized to analysis the target protein of Lut. RESULTS: PF effectively suppressed t-BHP-induced apoptosis caused by mitochondrial dysfunction, which were associated with inhibiting ROS generation. Further investigation indicated that PF significantly suppressed apoptosis, inflammation, and autophagy by regulating the expression of related proteins. The results of molecular docking and drug affinity targeting experiments (DARTS) revealed that PI3K was the target protein of PF and Lut. Further studies have shown that PF relieved liver injury induced by t-BHP via suppressing phosphorylated expression of PI3K. CONCLUSION: Our results indicate that PF effectively protect against hepatotoxicity induced by t-BHP through inhibiting the abnormal activation of PI3K-Akt signaling pathway and highlight the health benefits of PF regarding oxidative stress, proving it to be an important source of bioactive compounds associated with Nonalcoholic fatty liver disease (NAFLD).

Activation of hepatic CREBH and Insig signaling in the anti-hypertriglyceridemic mechanism of R-α-lipoic acid.

The activation of sterol regulatory element binding proteins (SREBPs) is regulated by insulin-induced genes 1 and 2 (Insig-1 and Insig-2) and SCAP. We previously reported that feeding R-α-lipoic acid (LA) to Zucker diabetic fatty (ZDF) rats improves severe hypertriglyceridemia. In this study, we investigated the role of cyclic AMP-responsive element binding protein H (CREBH) in the lipid-lowering mechanism of LA and its involvement in the SREBP-1c and Insig pathway. Incubation of McA cells with LA (0.2 mM) or glucose (6 mM) stimulated activation of CREBH. LA treatment further induced mRNA expression of Insig-1 and Insig-2a, but not Insig-2b, in glucose-treated cells. In vivo, feeding LA to obesity-induced hyperlipidemic ZDF rats activated hepatic CREBH and stimulated transcription and translation of Insig-1 and Insig-2a. Activation of CREBH and Insigs induced by LA suppressed processing of SREBP-1c precursor into nuclear SREBP-1c, which subsequently inhibited expression of genes involved in fatty acid synthesis, including FASN, ACC and SCD-1, and reduced triglyceride (TG) contents in both glucose-treated cells and ZDF rat livers. Additionally, LA treatment also decreased abundances of very low density lipoprotein (VLDL)-associated apolipoproteins, apoB100 and apoE, in glucose-treated cells and livers of ZDF rats, leading to decreased secretion of VLDL and improvement of hypertriglyceridemia. This study unveils a novel molecular mechanism whereby LA lowers TG via activation of hepatic CREBH and increased expression of Insig-1 and Insig-2a to inhibit de novo lipogenesis and VLDL secretion. These findings provide novel insight into the therapeutic potential of LA as an anti-hypertriglyceridemia dietary molecule.

Massage may initiate tendon structural changes--a preliminary study.

BACKGROUND: Physical exercise is now a widely known and studied factor of the proper functioning of living organisms. Many questions remain unanswered concerning various aspects of the changes in the morphology of structures subjected to chronic physical exercise. MATERIALS AND METHODS: This study was conducted on fifty Buffalo strain rats, randomly divided into two equal (experimental and control) groups. All animals were subjected to physical training on a running track for 10 weeks, whereas only in the experimental group, massage was additionally applied five-times per week. RESULTS: An increase in the percentage of collagen fibers in tendons with the smallest diameter (≤100 nm) was observed only in the experimental group in week 3, followed by a decrease in weeks 5 and 7. A subsequent repeated increase was observed in week 10 of the study. No significant differences were observed for either study group in the number of collagen fibers based on fiber diameter (101-200 nm, 201-300 nm and 301-400 nm). CONCLUSION: The results of this preliminary study showed that long-term massage performed during running training may initiate for small structural changes in the rat tendon. Further morphological studies with prolonged observation periods are recommended.

Protective effects of Akebia saponin D against rotenone-induced hepatic mitochondria dysfunction.

Akebia saponin D (ASD) is a typical bioactive triterpenoid saponin obtained from the rhizome of Dipsacus asper Wall. Previous studies have found that ASD has a hepatoprotective effect in a mouse model. The purpose of this paper was to explore the molecular mechanism of the hepatoprotective effects of ASD on BRL cells and isolated rat liver mitochondria. We investigated the effects of ASD on rotenone-induced toxicity in BRL cells. The results showed that ASD inhibited the accumulation of reactive oxidant species, ATP deficiency, and mitochondrial membrane potential dissipation; ameliorates mitochondrial respiratory dysfunction, and improved the activity of complex I in a concentration-dependent manner, indicating that ASD likely improved mitochondrial function. ASD suppressed rotenone-induced BRL cell apoptosis and increased Bcl-2/Bax ratio. These results suggest that ASD may exert hepatoprotective effects against rotenone-induced toxicity through mitochondria. This study supports our previous research that ASD possesses hepatoprotective activity in vivo and it is worthy of further study.

Increased metastatic potential of residual carcinoma after transarterial embolization in rat with McA-RH7777 hepatoma.

Transarterial chemoembolization represents a first-line non-curative therapy for hepatocellular carcinoma (HCC), although the biological changes in the remaining cancer after embolization are not completely understood. In the present study, we examined whether transarterial embolization (TAE) enhances the metastatic potential of residual HCC and investigated the mechanisms underlying embolization. The hepatoma cell line McA-RH7777, which is marked by green fluorescent protein (GFP), was used in the study. The invasion of cells cultured under hypoxia and normoxia was observed using the Transwell assay. Twenty male buffalo rats were implanted with GFP transfected McA-RH7777 tumors in the left lateral lobe of the liver. After laparotomy and retrograde placement of a catheter into the gastroduodenal artery (on the 14th day after implantation), TAE using lipiodol (0.2 ml/kg) was performed. Tumor volumes were measured before and after treatment using magnetic resonance imaging (MRI). Lung metastases were observed using fluorescence imaging, and the molecular changes of residual tumor cells were evaluated by western blotting or immunohistochemistry. The invasion assays indicated that the number of invading hypoxic cells was significantly higher than that of normoxic cells (30.2 ± 2.46 vs. 20.4 ± 1.89, P=0.013). Accompanying an increase in hypoxia-inducible factor-1α (HIF-1α) expression, the metastatic potential of tumor cells following hypoxia or TAE was enhanced. This enhanced metastatic potential was indicated by a significant reduction in the expression of E-cadherin and an upregulation of N-cadherin and vimentin expression. The number of lung metastases in the TAE group was 19.20 ± 1.76, whereas this number was 11.30 ± 1.54 in the control group, which represented a statistically significant difference (P=0.003). In conclusion, hypoxia in the residual tumor after TAE can increase the invasiveness and metastatic potential of HCC and may be responsible for the failure of TAE.

Herbal compound "Songyou Yin" attenuates hepatoma cell invasiveness and metastasis through downregulation of cytokines secreted by activated hepatic stellate cells.

BACKGROUND: Activated hepatic stellate cells (aHSCs) play an important role in the progression of hepatocellular carcinoma (HCC). Here, we determined if cytokines secreted in response to the herbal compound "Songyou Yin" (SYY) treatment of aHSCs could influence invasiveness and metastatic capabilities of hepatoma cells. METHODS: Primary rat hepatic stellate cells (HSCs) were isolated, activated, divided into SYY treated and untreated (nSYY) groups, and conditioned media (CM-SYY and CM-nSYY, respectively) were collected. The hepatoma cell line, McA-RH7777 was cultured for 4 weeks with SYY, CM-SYY, and CM-nSYY, designated McA-SYY, McA-SYYCM and McA-nSYYCM. The invasiveness and metastatic capabilities were evaluated using Matrigel invasion assay in vitro and pulmonary metastasis in vivo. Matrix metalloproteinase-2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and vimentin protein levels in McA-SYYCM and McA-nSYYCM were evaluated by Western blot. Cytokine levels in conditioned media were tested using enzyme-linked immunosorbent assay (ELISA). RESULTS: Matrigel invasion assay indicated that the number of McA-SYYCM cells passing through the basement membrane was less than in McA-nSYYCM cells (P < 0.01). Similar results were also observed in vivo for lung metastasis. McA-SYYCM cells showed less pulmonary metastasis capabilities than McA-nSYYCM cells (P < 0.001). The reduced expression of MMP-2 and reversed epithelial to mesenchymal transition with E-cadherin upregulation, and N-cadherin and vimentin downregulation were also found in McA-SYYCM compared to McA-nSYYCM. Metastasis-promoting cytokines hepatocyte growth factor, interleukin-6, transforming growth factor-ß1, and vascular endothelial growth factor were markedly decreased in CM-SYY compared to CM-nSYY. CONCLUSIONS: SYY attenuates hepatoma cell invasiveness and metastasis capabilities through downregulating cytokines secreted by activated hepatic stellate cells.

Hepatoprotection of 1ß-hydroxyeuscaphic acid - the major constituent from Rubus aleaefolius against CCl4-induced injury in hepatocytes cells.

CONTEXT: Rubus aleaefolius Poir. (Rosaceae) is used as a folk medicine to treat various types of hepatitis with significant effects in Fujian Province of China. The ethyl acetate fraction of R. aleaefolius root ethanol extract proved effective after our testing in vivo animal experiments. OBJECTIVE: The protective effects of a major constituent, 1ß-hydroxyeuscaphic acid isolated from R. aleaefolius was first investigated against carbon tetrachloride (CCl4)-induced injury in liver cells from hepatocytes cell line (BRL-3A). MATERIALS AND METHODS: Treatment of BRL-3A with CCl4 led to generation of free radicals detected after a 2 h incubation and produced cell injury demonstrated by increased leakage of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) in the media. Exposure to CCl4 caused apoptosis to cells but did not induce lipid peroxidation. Following treatment with 1ß-hydroxyeuscaphic acid at doses ranging from 1 to 100 µg/mL for 24 h, cellular morphology, cell growth function (MTT assay), ALT, AST, malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed and evaluated under control and exposed conditions. RESULTS: The IC50 of 1ß-hydroxyeuscaphic acid was 15 µg/mL. Exposure of injured BRL-3A at 20 µg/mL changed abnormal size, cellular shrinkage, and enhanced regulation. ALT, AST, MDA enzyme levels were reduced and SOD activity was increased. DISCUSSION AND CONCLUSION: Treatment with 1ß-hydroxyeuscaphic acid has significant hepatoprotective activity by lowering the leakage of intracellular enzymes, reducing the oxidation of proteins and decreasing the incidence of apoptosis. These results provide a basis for confirming the traditional uses of R. aleaefolius in treating hepatic diseases.

Effect of cellulose, pectin and chromium(III) on lipid and carbohydrate metabolism in rats.

The effects of cellulose, pectin and chromium(III) on lipid and carbohydrate metabolism were investigated in rats. Eighty male Buffalo rats (n=10/group, 4 weeks old) were fed experimental diets for 6 weeks. The two control groups received a fiber free diet (FF) or a fiber free diet plus chromium (FF+Cr) (2.53 mg Cr/kg diet). The other groups were fed diets containing 5% of cellulose (CEL), 5% of pectin (PEC) or 2.5% of cellulose plus 2.5% of pectin (CEL+PEC) with or without chromium. The daily food intake and body weight of the rats were not affected by the experimental diets. Total cholesterol level in plasma was significantly lower (p≤0.05) in the PEC group than the rats fed the FF diet. Feeding of rats with the PEC+Cr diet resulted in a significantly higher concentration of plasma HDL cholesterol (p≤0.05) when compared with the CEL+Cr group. No statistically significant differences in the concentrations of plasma triglycerides (TG) and non-esterified fatty acids (NEFA) between the groups were observed. Rats fed the CEL+Cr diet had a significantly lower content of cholesterol and rats fed the CEL+Cr diet lower contents of cholesterol and TG in the liver (p≤0.05) when compared with other groups. The concentration of HbA1c was significantly lower (p≤0.05) in rats fed the CEL and CEL+Cr diets than in other groups. A significantly lower concentration of plasma glucose (p≤0.05) was observed in rats receiving the CEL+PEC diet in comparison with the FF group. A significant effect of fiber and chromium combination was shown only in the case of triglyceride content in the liver of rats (p≤0.05). In conclusion, our results suggest that a diet containing fiber (PEC) and chromium or their supplements may be beneficial for correcting some disturbances of lipid metabolism, and a diet containing cellulose or its supplements may be used to improve glycemic control.

Hormonal, hypothalamic and striatal responses to reduced body weight gain are attenuated in anorectic rats bearing small tumors.

Lack of compensatory or even reduced food intake is frequently observed in weight-losing cancer patients and contributes to increased morbidity and mortality. Our previous work has shown increased transcription factor expression in the hypothalamus and ventral striatum of anorectic rats bearing small tumors. mRNA expression of molecules known to be involved in pathways regulating appetite in these structures was therefore assessed in this study. Given that pain, pro-inflammatory cytokines and metabolic hormones can modify food intake, spinal cord cellular activation patterns and plasma concentrations of cytokines and hormones were also studied. Morris hepatoma 7777 cells injected subcutaneously in Buffalo rats provoked a 10% lower body weight and 15% reduction in food intake compared to free-feeding tumor-free animals 4 weeks later when the tumor represented 1-2% of body mass. No differences in spinal cord activation patterns or plasma concentration of pro-inflammatory cytokines were observed between groups. However, the changes in plasma ghrelin and leptin concentrations found in food-restricted weight-matched rats in comparison to ad libitum-fed animals did not occur in anorectic tumor-bearing animals. Real-time PCR showed that tumor-bearing rats did not display the increase in hypothalamic agouti-related peptide mRNA observed in food-restricted weight-matched animals. In addition, microarray analysis and real-time PCR revealed increased ventral striatal prostaglandin D synthase expression in food-restricted animals compared to anorectic tumor-bearing rats. These findings indicate that blunted hypothalamic AgRP mRNA expression, probably as a consequence of relatively high leptin and low ghrelin concentrations, and reduced ventral striatal prostaglandin D synthesis play a role in maintaining cancer-associated anorexia.

Berberine reduces methylation of the MTTP promoter and alleviates fatty liver induced by a high-fat diet in rats.

High-calorie food leads to nonalcoholic fatty liver disease (NAFLD) through dysregulation of genes involved in lipid metabolism, but the precise mechanism remains unclear. DNA methylation represents one of the mechanisms that contributes to dysregulation of gene expression via interaction with environmental factors. Berberine can alleviate fatty liver in db/db and ob/ob mice. Here, we investigated whether DNA methylation is involved in the pathogenesis of NAFLD induced by a high-fat diet (HFD) and whether berberine improves NAFLD through influencing the methylation status of promoters of key genes. HFD markedly decreased the mRNA levels encoding CPT-1alpha, MTTP, and LDLR in the liver. In parallel, DNA methylation levels in the MTTP promoter of rats with NAFLD were elevated in the liver. Interestingly, berberine reversed the downregulated expression of these genes and selectively inhibited HFD-induced increase in the methylation of MTTP. Consistently, berberine increased hepatic triglyceride (TG) export and ameliorated HFD-induced fatty liver. Furthermore, a close negative correlation was observed between the MTTP expression and its DNA methylation (at sites -113 and -20). These data indicate that DNA methylation of the MTTP promoter likely contributes to its downregulation during HFD-induced NAFLD and, further, that berberine can partially counteract the HFD-elicited dysregulation of MTTP by reversing the methylation state of its promoter, leading to reduced hepatic fat content.

Thermoresponsive core-shell magnetic nanoparticles for combined modalities of cancer therapy.

Thermoresponsive polymer-coated magnetic nanoparticles loaded with anti-cancer drugs are of considerable interest for novel multi-modal cancer therapies. Such nanoparticles can be used for magnetic drug targeting followed by simultaneous hyperthermia and drug release. Gamma-Fe(2)O(3) iron oxide magnetic nanoparticles (MNP) with average sizes of 14, 19 and 43 nm were synthesized by high temperature decomposition. Composite magnetic nanoparticles (CNP) of 43 nm MNP coated with the thermoresponsive polymer poly-n-isopropylacrylamide (PNIPAM) were prepared by dispersion polymerization of n-isopropylacrylamide monomer in the presence of the MNP. In vitro drug release of doxorubicin-(dox) loaded dehydrated CNP at temperatures below and above the lower critical solution temperature of PNIPAM (34 degrees C) revealed a weak dependence of drug release on swelling behavior. The particles displayed Fickian diffusion release kinetics; the maximum dox release at 42 degrees C after 101 h was 41%. In vitro simultaneous hyperthermia and drug release of therapeutically relevant quantities of dox was achieved, 14.7% of loaded dox was released in 47 min at hyperthermia temperatures. In vivo magnetic targeting of dox-loaded CNP to hepatocellular carcinoma (HCC) in a buffalo rat model was studied by magnetic resonance imaging (MRI) and histology. In summary, the good in vitro and in vivo performance of the doxorubicin-loaded thermoresponsive polymer-coated magnetic nanoparticles suggests considerable promise for applications in multi-modal treatment of cancer.

Dietary carbohydrate content influences boron's effect on lipid and protein indices in rats.

The aim of the study was to establish whether an excess or deficiency of dietary carbohydrates would influence the effect of boron (B) on lipid, protein, and glucose metabolism in laboratory rats. Sixty male Buffalo rats were used in the study, divided into six groups fed a control diet or a lower high-carbohydrate diet with or without a B supplement (3 mg B/kg fodder). The hemoglobin concentration and hematocrit were assessed in whole-blood samples, and the total protein, albumin, creatine, glucose, total lipid, and high-density lipoprotein (HDL) cholesterol contents in the serum were established. The total cholesterol and triacylglycerol contents in liver lipid extracts were also measured. A low carbohydrate content in a B-supplemented diet led to an increase in the total protein and albumin contents in the serum of the rats compared to the levels for the rats on the control and high-carbohydrate B supplemented diets. Under conditions of an excess or deficiency of carbohydrates in the diet, B did not significantly influence the cholesterol and total lipid contents in the serum. Boron's influence on the other metabolic indexes (glucose, HDL cholesterol, and triacylglycerol contents in the serum; cholesterol and triacylglycerol contents in the liver) was unaffected by the carbohydrate content in the diet.

Influence of bioflavonoids from the radix extract of Scutellaria baicalensis on the level of serum lipids, and the development of laboratory rats fed with fresh and oxidized fats.

The aim of this study was to assess the influence of bioflavonoids from the radix of Scurellaria baicalensis on the level of lipids, via the determination of the concentrations of triglycerides, total cholesterol and high-density lipoprotein (HDL) cholesterol in the plasma of laboratory rats fed high-fat and high-cholesterol diets, and via the calculation of their atherogenic index. We also studied the influence of bioflavonoids on their physical development by measuring the increase in their body mass and liver mass. The rats were fed a diet with a 15% content of fresh or oxidized lard or sunflower oil, along with 0.5% added cholesterol. 0.05% S. baicalensis radix extract was added to the diet of half of the rats as the source of bioflavonoids. In the group of rats fed a diet containing oxidized oil we observed a significantly lower increase in body mass (15.5 +/- 7.6 g/4 weeks/rat) than that observed for the control rats (77.0 +/- 15.7 g/4 weeks/rat). The addition of S. baicalensis radix extract to the diet raised the increase in body mass in the groups receiving oil as the source of fat; those receiving fresh oil had a 40% increase, and those receiving oxidized oil showed a 300% increase relative to the appropriate controls. In all the groups of rats fed a diet with bioflavonoids added, a beneficial decrease in the plasma triglyceride content was observed relative to the appropriate controls. In the plasma of rats on a diet containing the extract and fresh or oxidized oil or fresh lard, we observed a beneficial reduction in the total cholesterol level relative to the appropriate controls. The atherogenic index was higher for the group of animals fed with fresh lard than for those fed with fresh oil, and higher for those fed with oxidized oil than for those fed with oxidized lard. The addition of bioflavonoids to the diet beneficially reduced the atherogenic index value in the group fed with fresh oil, and increased its value in the group fed with oxidized lard.

Low-frequency electromagnetic stimulation may lead to regression of Morris hepatoma in buffalo rats.

OBJECTIVE: The influence of low-frequency electromagnetic (LF-EM) waves on the processes of carcinogenesis and tumor growth has been the subject of experimental investigations for more than two decades and the results are promising. In parallel, an interesting method of complementary medicine, biophysical-information therapy (BIT) or bioresonance therapy (BRT), which in principle is based on LF-EM stimulation, has emerged. BRT has been known since the late 1980s but is still poorly studied. The idea of applying BRT to tumors is based on two main premises: (1) endogenous biophotonic emission in the case of tumors is different from that produced by healthy tissues/cells and (2) BRT effects are believed to be primarily manifested at the immune-system level. Consequently, we decided to study the influence of BRT on a dynamic and well-known process: the expansion of transplantable hepatoma in rats. DESIGN: The study was carried out on female Buffalo rats with implanted Morris tumors (three experimental and one control group). Fourteen (14) consecutive in vivo exposures using a BRT device (BICOM B15, REGUMED Regulative Medizintechnik Gmbh, Graefelfing, Germany), were made from the third day after inoculation of the tumors. Biometric observations, intra vitam (tumor volume, growth ratio), were completed with histologic investigation (implantation locus, selected internal organs [lungs]). RESULTS: Thirty-one (31) cases (69%; n = 45) of total tumor regression were observed in experimental groups and these individuals were anesthetized to enable histologic verification to be made. No lung metastases--usually observed in tumor-bearers--could be detected. Moreover, in the inoculation loci, traces of former implantation and tumor absorption were found to be associated with high activity of cell-mediated immune response. No regressions were observed in the control group. CONCLUSIONS: We cannot exclude the possibility that LF-EM signals transmitted via BRT into the tumor-bearers may stimulate two separate processes: effective immunological response and/or tumor-cell death. The method appears to be capable of inducing the regression of transplantable hepatoma in vivo, thus is a potential subject of further studies.

Melatonin uptake and growth prevention in rat hepatoma 7288CTC in response to dietary melatonin: melatonin receptor-mediated inhibition of tumor linoleic acid metabolism to the growth signaling molecule 13-hydroxyoctadecadienoic acid and the potential role of phytomelatonin.

Both physiological and pharmacological levels of the pineal hormone melatonin exhibit substantial anticancer activity in tissue-isolated rat hepatoma 7288CTC via melatonin receptor-mediated blockade of tumor uptake of linoleic acid (LA) and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). Melatonin is also present in significant amounts in edible plants and is supplied in nutritional supplements. We confirmed the presence of significant quantities of melatonin in 20 varieties of edible plants. In pinealectomized tumor-free rats, 3 weeks of ingestion of either 5 or 50 microg/day of melatonin contained in a semi-purified diet resulted in a dose-dependent elevation in steady-state plasma melatonin levels within the nocturnal physiological range. In pineal-intact tumor-bearing rats, the daily intake of 5 microg/day of melatonin for 3 weeks resulted in an enhanced amplitude and duration of the nocturnal melatonin levels within physiological circulating limits. The nocturnal melatonin amplitude in rats ingesting 500 ng of melatonin/day remained within the physiological range. A dose-related increase in tumor concentrations of melatonin occurred in animals ingesting melatonin from the diet. Perfusion of tumors in situ with physiological, nocturnal blood levels of melatonin resulted in a mean 31% uptake and retention of the melatonin. Chronic ingestion of 50 ng, 500 ng or 5 microg of melatonin/day supplied in a semi-purified 5% corn oil diet led to a significant dose-dependent reduction in the rates of tumor total fatty acid uptake, LA uptake, 13-HODE production and tumor growth. The co-ingestion of melatonin receptor antagonist S20928 completely blocked the effects and prevented the intra-tumoral accumulation of melatonin. Melatonin receptor-mediated suppression of tumor growth, LA uptake and metabolism, and stimulation of tumor melatonin uptake and retention in response to the dietary intake of phytomelatonin from edible plants or melatonin from nutritional supplements, could play an important role in cancer growth prevention.

Sensitivity to drug effects on prepulse inhibition in inbred and outbred rat strains.

Genetic differences in the neurochemical regulation of PPI in rats may help clarify the neural basis of inherited PPI differences in neuropsychiatric disorders. We reported and characterized substantial heritable differences in sensitivity to PPI-disruptive effects of DA agonists in outbred Sprague Dawley (SDH) versus Long-Evans (LEH) rats. Other strains might yield large group separations and facilitate studies of the neural basis for these strain differences; inbred strains might also allow us to map genes associated with differential PPI sensitivity. Sensitivity to the PPI-disruptive effects of the DA agonist apomorphine (APO) and the NMDA antagonist phencyclidine (PCP) were compared across inbred and outbred strains. APO sensitivity was greatest in SDH and buffalo rats, but the effect in buffalo rats was complicated by significant APO-induced startle suppression. PPI APO sensitivity was least in ACI and LEH rats; F344s exhibited intermediate sensitivity and Lewis rats showed a nonlinear dose response (sensitivity at low but not higher doses). PPI APO insensitivity in ACI rats developed over time, with ACI pups exhibiting robust sensitivity. Substantial strain differences were observed in short-interval (10-30 ms) prepulse effects, and APO-induced increases in short-interval PPI occurred in SDH, LEH, and Lewis rats, but not in F344, ACI, or buffalo rats. Sensitivity to PPI-disruptive effects of PCP was generally greater in outbred than inbred rats. These findings identify strains suitable for comparisons of PPI neural circuitry and others for whom such comparisons would be complex and perhaps less informative.

Curcumin enhances the immunosuppressive activity of cyclosporine in rat cardiac allografts and in mixed lymphocyte reactions.

Curcumin (CCM; diferuoylmethane) is a dietary pigment in curry with known antineoplastic and anti-inflammatory effects. The immunosuppressive effects of CCM were studied in (1) rat heterotopic cardiac transplant models, using Brown-Norway (BN, RT1(n)) hearts to WKY (RT1(u)) hosts or Buffalo (BUF, RT1(b)) hearts to Wistar-Furth (WF, RT1(u)) hosts, (2) reverse transcriptase-polymerase chain reaction analysis of cytokines from transplanted specimens, and (3) mixed lymphocyte reactions (MLR). In the BN-to-WKY model, CCM alone significantly increased the mean survival time (MST) to 20.5 to 24.5 days, as compared to 9.1 days among nontreated controls. The combination of CCM and subtherapeutic doses of CsA produced further prolongation of the MST to 28.5 to 35.6 days, better than that of CCM or CsA alone (P <.05). In a BUF-to-WF model, CCM alone did not increased the MST, unless it was combined with subtherapeutic doses of CsA, wherein two thirds of the grafts survived for more than 60 days (P <.05 as compared to either treatment group). Cytokine analysis revealed significantly reduced expression of interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and granzyme B in the day 3 specimens of the CCM and CCM CsA-treated allografts compared with the nontreated allograft controls. MLRs using the two MHC-incompatible rat strains (BNxWKY) showed an effect of increasing concentrations of CCM and/or CsA, which by combination index (CI) analysis showed a synergistic effect (CI = 0.22 to 0.81). This study for the first time demonstrates the effectiveness of CCM as a novel adjuvant immunosuppressant with cyclosporine both in vivo and in vitro.

Evodiamine, a constituent of Evodiae Fructus, induces anti-proliferating effects in tumor cells.

We found that evodiamine, a major alkaloidal component of Evodiae Fructus (Goshuyu in Japan), inhibited proliferation of several tumor cell lines, but had less effect on human peripheral blood mononuclear cells (PBMC). We used human cervical cancer cells, HeLa, as a model to elucidate the molecular mechanisms of evodiamine-induced tumor cell death. The results showed that evodiamine induced oligonucleosomal fragmentation of DNA in HeLa cells and increased the activity of caspase-3, but not that of caspase-1, in vitro. Both evodiamine-induced DNA fragmentation and caspase-3 activity were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). In addition, evodiamine increased the expression of the apoptosis inducer Bax, but decreased the expression of the apoptosis suppressor Bcl-2 in mitochondria. Taken together, our data indicated that evodiamine alters the balance of Bcl-2 and Bax gene expression and induces apoptosis through the caspase pathway in HeLa cells.