Electroacupuncture enhances hippocampal NSCs proliferation in cerebral ischemia-reperfusion injured rats via activation of notch signaling pathway.

The purpose of this study was to investigate the mechanisms by which electroacupuncture (EA) enhances hippocampal neural stem cells (NSCs) proliferation in cerebral ischemia-reperfusion (I/R) injured rats. A total of 72 male adult Sprague-Dawley rats were randomly divided into the sham operation control group (SC), the ischemia control group (IC) and the EA group. Middle cerebral artery occlusion (MCAO) was performed to establish the focal cerebral I/R injury model. Proliferation of hippocampal NSCs in cerebral I/R injured rats was determined by the Nestin immunohistochemical staining. Activation of the notch signaling pathway was detected by Western blotting and reverse transcription polymerase chain reaction analysis. The serum level of neurotrophic factors, e.g., the brain-derived neurotrophic factor (BDNF) and the Glial cell line-derived neurotrophic factor (GDNF), was measured using enzyme-linked immunosorbent assay (ELISA). The results showed that EA at Quchi (LI11) and Zusanli (ST36) acupoints significantly alleviated neurological deficits, reduced infarct volumes and promoted the proliferation of hippocampal NSCs in cerebral I/R injured rats. The crucial signaling molecules in the notch signaling pathway were activated and the secretion of BDNF and GDNF was increased upon EA. The protein and mRNA levels of Cyclin D1, Cdk4 and p-Rb were increased, while p21 and p27 transcripts were suppressed by notch signaling. These results suggest that the up-regulatory effect of EA on the notch signaling pathway and neurotrophic factor secretion may result in the promotion of NSCs proliferation and consequently a therapeutic effect on cerebral ischemia.

[A system review of randomized controlled trials on treating chronic stable angina by rhodiola].

OBJECTIVE: To systematically assess the efficacy and safety of Rhodiola in treating chronic stable angina pectoris. METHODS: Our group searched the Cochrane library, PubMed, Embase, Chinese biomedical literature database (CBM), VIP database (VIP), Chinese Journal Full-text Database (CNKI) for the literature published in English and Chinese till April 2013. Randomized controlled trials (RCTs) were included on the therapeutic effect of Rhodiola or Rhodiola plus conventional Western medicine in comparison with the conventional Western medicine treatment on stable angina. Data were extracted according the data extraction form. The literature methodological quality was assessed by using the Cochrane handbook, and data analyzed by Rev-Man 5.2 Software for Meta-analysis. The effect indicators of outcomes was expressed by odds ratio (OR) and 95% CI. RESULTS: A total of 7 randomized controlled trials, 662 cases of stable angina pectoris patients met the inclusion criteria and all published in Chinese, without one scientific design and high quality literature. Compared with the conventional Western medicine treatment, combined with oral administration of Rhodiola could improve the efficiency of anti-angina (OR = 2.49, 95% CI: 1.02 - 6.09). Combined with intravenous infusion of Rhodiola could also improve the efficacy of angina pectoris (OR = 4.86, 95% CI: 2.4 - 9.82). Oral administration of Rhodiola couldn't improve ECG efficacy (OR = 1.25, 95% CI: 0.67 - 2.34). Intravenous infusion of Rhodiola could improve the clinical efficacy (OR = 2.94, 95% CI: 1.61 - 5.35). Combined with the conventional treatment, intravenous infusion of Rhodiola could improve the whole blood viscosity (low and high shear rates) and inverse variance (IV) (-1.36 and -0.99, 95% CI: -1.65 - 1.07 and -1.26 - 0.71), but could not reduce serum fibrinogen and D-dimer level. The incidence rate of adverse reactions was higher than that of the conventional treatment combined with Rhodiola (OR = 0.1, 95% CI: 0.02 - 0.51). CONCLUSIONS: On the basis of routine treatment, Rhodiola could further improve patients' symptoms. Combined with intravenous medication, Rhodiola could increase the ECG improvement rate, and reduce adverse reactions. But the methodological quality of included studies was poor, the number of samples was small, and influence factors such as the intervention period was short. This conclusion needs scientific and rational design in a larger sample, multicenter clinical trial to verify.

[Effect of bear bile powder on STAT3 pathway in hepatocellular carcinoma xenograft].

OBJECTIVE: To observe the effect of bear bile powder (BBP) on the STAT3 pathway and its downstream target genes of nude mice hepatocellular carcinoma (HCC) xenograft, and to explore its mechanism for treating HCC. METHODS: The subcutaneous xenograft model was established using HepG2 cells. When the subcutaneous transplanted tumor was formed, naked mice were randomly divided into two groups, the BBP group and the control group. Mice in the BBP group were administered with BBP by gastrogavage, once daily for 3 consecutive weeks, while mice in the control group were administered with normal saline by gastrogavage, once daily for 3 consecutive weeks. The body weight and the tumor volume were measured once per week. By the end of medication, the tumor weight was weighed and the tumor inhibition ratio calculated. The apoptosis of the tumor tissue was detected by TdT-mediated dUTP nick end labeling (TUNEL). The expression of Bcl2-associated X protein (Bax), B cell lymphoma/eukemina-2 (Bcl-2), cyclin-dependent protein kinase (CDK4), cyclinD1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression levels of signal transducers and transcription activators 3 (p-STAT3), proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, CDK4, and cyclinD1 were determined by immunohistochemistry. RESULTS: BBP could inhibit the tumor volume and tumor weight, showing statistical difference when compared with the control group (P < 0.01). Results of TUNEL showed that BBP could significantly induce the apoptosis of hepatoma carcinoma cells. Results of RT-PCR showed that BBP could up-regulate the expression of Bax and down-regulate mRNA expression of Bcl-2, CDK4, and cyclinD1. Immunohistochemical results showed that BBP could up-regulate the expression of Bax and inhibit the protein expression of p-STAT3, PCNA, Bcl-2, CDK4, and cyclinD1. CONCLUSION: BBP could induce the apoptosis of hepatoma carcinoma cells and inhibit their proliferation by regulating STAT3 pathway.

Rapid simultaneous determination of twelve major components in Pien Tze Huang by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry.

An efficient method using ultra-performance LC coupled with triple quadrupole MS was developed for the rapid determination of 12 major active components in Pien Tze Huang (PZH), a well-known traditional Chinese formula. Chromatographic separation was achieved on a Waters XBridge BEH RP18 column (50 mm × 2.1 mm id, 1.7 µm) with a gradient mobile phase (A: 0.1% aqueous formic acid and B: acetonitrile with 0.1% formic acid) at a flow rate of 0.8 mL/min. The chromatographic peaks of 12 components were identified by comparing their retention time and MS data with the related reference compounds. Multiple-reaction monitoring was employed for the quantitative analysis. Ten batches of PZH were analyzed with a good linear regression relationship (r, 0.9987­0.9995), intraday precisions (RSD, 2.05­4.80%), interday precisions (RSD, 1.99­4.98%), repeatability (RSD, 2.21­4.20%), stability (RSD, 3.52­4.81%), and recovery (95.63­104.80%). By using this established method, the present study offered highly sensitive, specific, and speedy determination of 12 major components, which promoted the quality control investigation of PZH greatly.

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.

Scutellaria barbata D. Don inhibits tumor angiogenesis via suppression of Hedgehog pathway in a mouse model of colorectal cancer.

Angiogenesis, which plays a critical role during tumor development, is tightly regulated by the Sonic Hedgehog (SHH) pathway, which has been known to malfunction in many types of cancer. Therefore, inhibition of angiogenesis via modulation of the SHH signaling pathway has become very attractive for cancer chemotherapy. Scutellaria barbata D. Don (SB) has long been used in China to treat various cancers including colorectal cancer (CRC). Our published data suggested that the ethanol extract of SB (EESB) is able to induce apoptosis of colon cancer cells and inhibit angiogenesis in a chick embryo chorioallantoic membrane model. To further elucidate the precise mechanisms of its anti-tumor activity, in the present study we used a CRC mouse xenograft model to evaluate the effect of EESB on tumor growth and angiogenesis in vivo. Our current data indicated that EESB reduces tumor size without affecting on the body weight gain in CRC mice. In addition, EESB treatment suppresses the expression of key mediators of the SHH pathway in tumor tissues, which in turn resulted in the inhibition of tumor angiogenesis. Furthermore, EESB treatment inhibits the expression of vascular endothelial growth factor A (VEGF-A), an important target gene of SHH signaling and functioning as one of the strongest stimulators of angiogenesis. Our findings suggest that inhibition of tumor angiogenesis via suppression of the SHH pathway might be one of the mechanisms by which Scutellaria barbata D. Don can be effective in the treatment of cancers.

Hedyotis diffusa Willd inhibits colorectal cancer growth in vivo via inhibition of STAT3 signaling pathway.

Signal Transducer and Activator of Transcription 3 (STAT3), a common oncogenic mediator, is constitutively activated in many types of human cancers; therefore it is a major focus in the development of novel anti-cancer agents. Hedyotis diffusa Willd has been used as a major component in several Chinese medicine formulas for the clinical treatment of colorectal cancer (CRC). However, the precise mechanism of its anti-tumor activity remains largely unclear. Using a CRC mouse xenograft model, in the present study we evaluated the effect of the ethanol extract of Hedyotis diffusa Willd (EEHDW) on tumor growth in vivo and investigated the underlying molecular mechanisms. We found that EEHDW reduced tumor volume and tumor weight, but had no effect on body weight gain in CRC mice, demonstrating that EEHDW can inhibit CRC growth in vivo without apparent adverse effect. In addition, EEHDW treatment suppressed STAT3 phosphorylation in tumor tissues, which in turn resulted in the promotion of cancer cell apoptosis and inhibition of proliferation. Moreover, EEHDW treatment altered the expression pattern of several important target genes of the STAT3 signaling pathway, i.e., decreased expression of Cyclin D1, CDK4 and Bcl-2 as well as up-regulated p21 and Bax. These results suggest that suppression of the STAT3 pathway might be one of the mechanisms by which EEHDW treats colorectal cancer.

[Visual acupotomy intervention mitigates pain reaction by improving intervertebral disc degeneration and inhibiting apoptosis of nucleus pulposus cells in rabbits with cervical spondylosis].

OBJECTIVE: To investigate the effect of visual acupotomy intervention on intervertebral disc degeneration, nucleus pulposus cell apoptosis and expression of apoptosis related proteins in rabbits with cervical spondylosis (CS), so as to explore its mechanism underlying improvement of CS. METHODS: A total of 48 male New Zealand rabbits were randomly divided into blank control, model, acupotomy and medication (meloxicam) groups, with 12 rabbits in each group. The neck type CS model was established by forcing the rabbit to make a neck flexion for 5 hours in a restrained chamber, once daily for 12 weeks. Rabbits of the medication group received an intramuscular injection of meloxicam (0.35 mg/kg), once daily for 4 consecutive weeks, and those of the acupotomy group received ultrasound-guided acupotomy intervention, once a week for 4 weeks. The pain threshold (PT) was measured by using a VonFrey electronic pain detector. The levels of prostaglandin E2 (PGE2), 5-hydroxytryptamine (5-HT) and substance P (SP) in serum were detected by ELISA. The severity of intervertebral disc degeneration was observed by using magnetic resonance imaging (MRI) and given scores in accordance with Suzuki's and colleague's "new classification system of cervical disk degeneration". The apoptosis of nucleus pulposus cells was analyzed by TUNEL staining. The protein expression levels of apoptosis-related protein Fas, cysteinyl aspartate-specific protease-3 (Caspase-3), B-cell lymphoma-2 asso-ciated X protein (Bax) and B-cell lymphoma-2 protein (Bcl-2) were measured by Western blot. RESULTS: Compared with the blank control group, the PT and Bcl-2 expression and MRI score were significantly down-regulated (P<0.01, P<0.001), whereas the contents of serum PGE2, 5-HT and SP, ratios of TUNEL-positive cells, and expression of Fas, Caspase-3 and Bax were considerably up-regulated (P<0.001, P<0.05, P<0.01) in the model group. In contrast to the model group, both the medication and acupotomy groups had an obvious increase in the levels of PT and Bcl-2 expression and MRI score (P<0.05, P<0.01), and a significant decrease in the contents of serum PGE2, 5-HT, SP, ratios of TUNEL-positive cells, and expression of Fas, Caspase-3 and Bax proteins (P<0.05). No significant differences were found between the medication and acupotomy groups in all the indexes mentioned above (P>0.05). CONCLUSION: Visual acupotomy intervention can mitigate the pain state of CS rabbits, which may be related to its functions in improving the intervertebral disc degeneration, reducing inflammatory reactions and apoptosis of nucleus pulposus cells.

Hepatoprotection in a rat model of acute liver damage through inhibition of CY2E1 activity by total alkaloids extracted from Rubus alceifolius Poir.

We aimed to examine the effect of an alkaloid extract of the roots of Rubus alceifolius Poir on liver damage and cytochrome enzymes, and underlying mechanism. Hepatotoxicity was induced in rats by treatment with carbon tetrachloride (CCl(4)). Rats were then treated with the hepatoprotective drug bifendate, or with low, medium, and high doses of an alkaloid extract from the roots of R alceifolius Poir. Both bifendate and alkaloid treatment decreased the increase in liver enzymes and cell damage caused by CCl(4). Carbon tetrachloride treatment alone caused a decrease in total cytochrome P450 content, an increase in CYP2E1 and CYP3A1 messenger RNA (mRNA) levels, and an increase in CYP2E1 and a decrease in CYP3A1 enzymatic activity. Alkaloid treatment brought these concentrations and activities back toward normal. In summary, these results suggest that alkaloids from R alceifolius Poir may act to protect the liver through decreasing CYP2E1 enzymatic activity through decreasing its mRNA.

[Effect of total alkaloids of Rubus alceaefolius on oxidative stress in rats with non-alcoholic fatty liver disease].

OBJECTIVE: To study the effects of total alkaloids of Rubus alceaefolius (RAP) on non-alcoholic fatty liver disease (NAFLD) rats and explore its possible mechanisms. METHOD: Sixty SD rats were randomly divided into six groups: control group, model group, compound methionine and choline bitartrate tablets (CMCB)group and three RAP groups treated respectively with low, middle and high dose of RAP. The NAFLD model was induced by feeding fat-rich food. NAFLD rats were administrated with 0.35 g x kg(-1) CMCB and 0.36, 0.72, 1.44 g x kg(-1) RAP for 4 weeks respectively. The weight index of liver was measured. Hepatic histolog ical changes were observed. The concentration in serum of aspartate amino transferase (AST), alanine amino tranferase (ALT), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were determined. The mRNA expressions of SOD, MDA, TNF-alpha and IL-6 in hepatic tissue were detected. RESULT: Compared with the model group, degree of steatosis of hepatic lobule was improved, the weight index of liver was decreased, serum levels of ALT, AST, TNF-alpha and IL-6 were significantly lower in the high and middle dose RAP group (P < 0.05 or P < 0.01). The levels of SOD and MDA in hepatic tissue were lower in the high dose RAP group (P < 0.05). The mRNA expressions of TNF-alpha and IL-6 in hepatic tissue were decreased (P < 0.05 or P < 0.01). CONCLUSION: RAP can protect liver in experimental NAFLD, and its possible mechanisms may be concerned with clearing the oxygen free radical, reducing the product of lipid peroxidation, inhibiting the release of inflammatory cytokines and reducing nflammatory response.

[Ultrastructure and TUNEL staining on inhibition of Rubus alceaefolius total alkaloids for apoptosis of liver in rat models of acute hepatitis].

OBJECTIVE: To investigate the anti-apoptosis effects of Rubus alceaefolius total alkaloids in rats with hepatic injury. METHOD: The hepatic injury model of rat was induced by intraperitoneal injection with CCl4. Sixty SD rats were randomly divided into the normal group, the model group, the R. alceaefolius total alkaloids intervened group, and the bifendate intervened group. The expressions of the levels of liver cell apoptosis were determined by TUNEL. Ultrastructures of the liver cells were observed with transmission electron microscope. RESULT: Compared with the model group, the degree of hepatic injury and the positive expressions of apoptosis in liver tissues in the R. alceaefolius total alkaloids intervened groups and the bifendate intervened group were significantly lower. CONCLUSION: R. alceaefolius total alkaloids could reduce the pathological changes and degree of hepatic injury in rats, which may be partially through inhibiting the expressions of apoptosis in liver tissue.

Cofilin Inhibition by Limk1 Reduces Rod Formation and Cell Apoptosis after Ischemic Stroke.

Cofilin, a cytoskeletal actin severing protein, is essential for the initiation phase of apoptosis. The formation of cofilin rods (containing 1:1 cofilin:actin) has been studied in cultured mammalian neurons under conditions of excessive glutamate, ATP depletion (ATP-D) or oxidative stress. These conditions simulate the pathologies occurring during ischemic stroke. In this study, we investigated the potential involvement of cofilin during ischemic-stroke induced apoptosis. Transient middle cerebral artery occlusion (tMCAO) was performed to establish an experimental model of ischemic stroke. We used 2,3,5-Triphenyltetrazolium Chloride (TTC) and immunostaining of the neuronal marker neuronal nuclei (NeuN) to evaluate the evolving phases of infarction in rats subjected tMCAO. Immunostaining and TdT-mediated dUTP Nick-End Labeling (TUNEL) apoptosis staining were collaboratively used to examine cofilin rod formation and cell apoptosis in response to ischemia at different time points (2 h, 8 h, 24 h and 7 d). Our results showed that infarct volume increased initially, between the first 2 h to 24 h and became stabilized 24 h to 7 d after tMCAO. The formation of cofilin rods significantly increased in the cortical core (from 2 h) and penumbra (from 8 h), peaking at 24 h and gradually diminishing 7 d after tMCAO. Progressive accumulation of cofilin rods subsequently induced microtubule-associated protein-2 (MAP2) degradation and ischemic cell apoptosis in the infarct cortex after stroke. To further corroborate the role of activated cofilin in ischemic stroke, inhibition of cofilin by LIM kinase (Limk1) over-expression was performed. Lmik1 reduced cofilin rod formation and MAP2 degradation, and consequently, attenuated cofilin mediated-apoptosis 24 h after tMCAO. From this evidence we conclude that cofilin plays a role in the onset of ischemic-induced apoptosis and may be efficacious in future studies as a drug target for ischemic stroke.

[Effect of total alkaloids of Rubus alceaefolius poiron on gene expressions of CYP2E1 and CYP3A1 in rats with acute liver injury].

OBJECTIVE: To explore the effects of total alkaloids of Rubus alceaefolius Poiron (RAP) on gene expressions of drug-metabolic enzymes, CYP2E1 and CYP3A1 in liver. METHODS: Sixty SD rats were randomly divided into six groups (10 rats in each), the blank control group, the model control group, the bifendate group and the three RAP treated groups treated respectively with low-, middle- and high-dose of RAP. The model of acute hepatic injury was established with intra-peritoneal injection of carbon tetrachloride. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and severity of hepatic tissue injury were measured, and the mRNA expressions of CYP2E1 and CYP3A1 in liver tissue were detected by RT-PCR. RESULTS: As compared with the model group, serum levels of ALT and AST were significantly lower in the high- and middle-dose ARP group (P <0.01), but in the low-dose group, only ALT was significantly lower (P<0.01); the severity of liver injury was milder in the RAP groups (P<0.01); and both CYP2E1 and CYP3A1 mRNA expressions in liver were significantly lower in the bifendate and all RAP treated groups (P<0.01 or P<0.05). CONCLUSION: RAP could significantly reduce the ALT and AST levels, protect liver cells from injury, and inhibit the mRNA expressions of CYP2E1 and CYP3A1 in liver tissue.

[Effect of kangquan recipe on basic fibroblast growth factor of prostate tissue in rats].

OBJECTIVE: To investigate the effect of Kangquan Recipe (KQR) on basic fibroblast growth factor (bFGF) in prostatic tissue of rats. METHODS: Benign prostatic hyperplasia model rat was established by injecting testosterone after castration. After being administered with KQR by gastrogavage for 30 days, the model rats were killed and their abdominal lobe prostate glands were taken for determining the protein and mRNA expressions of bFGF using immunohistochemical method and RT-polymerase chain reaction (RT-PCR) respectively. RESULTS: The expressions of bFGF and bFGF mRNA were significantly lower in the model rats being treated with high or medium dose of KQR than those in the untreated model rats (P < 0.05 or P < 0.01), but these indexes were insignificantly different between the model rats treated with low dose of KQR and untreated model rats (P > 0.05). CONCLUSION: KQR can effectively decrease the expression of bFGF in prostatic tissue of experimental benign prostate hyperplasia model rats.

[Candidate targets for research on benign prostatic hyperplasia].

The etiology and pathogenesis of benign prostatic hyperplasia are very complicated, about which a variety of theories have been developed, so it is of utmost importance to decide upon the target of research. Focusing on the pathogenesis of benign prostatic hy-perplasia, the author outlines the candidate targets for the experimental studies of the disease in such approaches as morphology, hormones, growth factors and genes.

[Effect of Kangquan Recipe on apoptosis regulatory genes bax and bcl-2 mRNA in prostate of rats].

OBJECTIVE: To investigate the effect of Kangquan Recipe (KQR) on apoptosis regulatory genes bax and bcl-2 mRNA in prostate of rats. METHODS: Benign prostatic hyperplasia model rat was established by injecting testosterone after castration. The model rats were killed and prostate glands were removed for examination after being treated with administration of KQR by gastrogavage for 30 days. The wet weight of prostate was measured and the mRNA expressions of bax and bcl-2 in rats' tissue of abdominal lobe of prostate were determined by RT-PCR. RESULTS: Compared with the model group, wet weight of prostate was lower significantly in the groups treated with different dosages of KQR (P < 0.05 or P < 0.01), and that in the high dose KQR treated group was similar to that in the normal group (P > 0.05). Compared with the model group, the expressions of bax mRNA and ratios of bax/bcl-2 were significantly higher and the expressions of bcl-2 mRNA significantly lower in the KQR treated groups (P < 0.01), and these indexes in the high dose KQR treated group were insignificantly different from those in the normal group (P > 0.05). CONCLUSION: KQR shows an obvious treatment effect on rats with benign prostatic hyperplasia, the mechanism might be through effectively regulating the expressions of bax mRNA and bcl-2 mRNA in prostatic tissue to accelerate the cell apoptosis of prostate in obvious dose-effect manner.

Ethanol extract of Antrodia camphorata inhibits proliferation of HCT-8 human colorectal cancer cells by arresting cell cycle progression and inducing apoptosis.

Antrodia camphorata (AC) is well known in Taiwan as a traditional Chinese medicine, with a long history of use in treating cancer and inflammation. Previous studies have revealed that AC exhibits anticancer effects in various cancer cell lines. However, the inhibitory influence of AC on colorectal cancer (CRC) cell growth and survival remains unknown. The present study investigated the effects of AC on the proliferation, survival, and cell cycle­ and apoptosis­associated gene and protein expression in the HCT­8 human CRC cell line in vitro. The antitumor activity of AC against HCT­8 cells was assessed using cell viability and colony formation assays. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis and morphological alterations were assessed by Hoechst 33258 staining and microscopy. The mRNA expression of cell cycle­ and apoptosis­associated genes was determined by reverse transcription­quantitative polymerase chain reaction, and protein expression levels of B­cell lymphoma 2 (Bcl­2), Bcl­2 X associated protein (Bax) cyclin D1, cyclin dependent kinase 4 (CDK4) and MYC proto­oncogene bHLH transcription factor (c­Myc) were determined by western blotting. Treatment of HCT­8 cells with various concentrations of AC (0.4­1.2 mg/ml) resulted in dose­ and time­dependent reductions in cell viability. HCT­8 cell cycle was arrested in the G0/G1 phase or G0/G1 and G2/M phases following AC treatment, compared with untreated cells. Furthermore, AC markedly inhibited HCT­8 cell growth with induction of apoptotic alterations and inhibition of proliferation. AC treatment induced HCT­8 cell apoptosis, upregulated expression of the apoptosis gene Bax, and downregulated Bcl­2, cMyc, cyclin D1 and CDK4 protein expression levels. The present data demonstrated that AC exhibited antiproliferative and growth inhibition effects on HCT­8 cells via induction of apoptosis and blocking of cell cycle progression, thus suggesting that it may have anticancer properties valuable for potential future therapeutic application for the treatment of CRC.

THE HERBAL MIXTURE XIAO-CHAI-HU TANG (XCHT) INDUCES APOPTOSIS OF HUMAN HEPATOCELLULAR CARCINOMA HUH7 CELLS IN VITRO AND IN VIVO.

BACKGROUND: Xiao-Chai-Hu Tang (XCHT) is an extract of seven herbs with anticancer properties, but its mechanism of action is unknown. In this study, we evaluated XCHT-treated hepatocellular carcinoma (HCC) for anti-proliferative and pro-apoptotic effects. MATERIALS AND METHODS: Using a hepatic cancer xenograft model, we investigated the in vivo efficacy of XCHT against tumor growth by evaluating tumor volume and weight, as well as measuring apoptosis and cellular proliferation within the tumor. To study the effects of XCHT in vitro, we measured the cell viability of XCHT-treated Huh7 cells, as well as colony formation and apoptosis. To identify a potential mechanism of action, the gene and protein expression levels of Bax, Bcl-2, CDK4 and cyclin-D1 were measured in XCHT-treated Huh7 cells. RESULTS: We found that XCHT reduced tumor size and weight, as well as significantly decreased cell viability both in vivo and in vitro. XCHT suppressed the expression of the proliferation marker Ki-67 in HCC tissues and inhibited Huh7 colony formation. XCHT induced apoptosis in HCC tumor tissues and in Huh7 cells. Finally, XCHT altered the expression of Bax, Bcl-2, CDK4 and cyclin-D1, which halted cell proliferation and promoted apoptosis. CONCLUSION: Our data suggest that XCHT enhances expression of pro-apoptotic pathways, resulting in potent anticancer activity.

Anti-angiogenic effect of Livistona chinensis seed extract in vitro and in vivo.

The present study aimed to detect the impact of the ethanol extract of the Livistona chinensis seed (EELC) on angiogenesis in human umbilical vein endothelial cells (HUVECs). A chorioallantoic membrane (CAM) assay was used to detect the anti-angiogenic activity of EELC in vivo. In vitro, the effect of EELC on the proliferation, migration and angiogenesis of HUVECs was determined by an MTT assay, a wound healing assay and a tube formation assay, respectively. The vascular endothelial growth factor (VEGF)-A and VEGF receptor (VEGFR)-2 protein and mRNA level were measured with ELISA and reverse transcription-semi-quantitative polymerase chain reaction. It was observed that EELC significantly decreased the formation of new vessels in the CAM assay. EELC inhibited the proliferation and migration of HUVECs. The extent of tube formation by HUVECs was also reduced by EELC. In addition, EELC treatment reduced the level of VEGF-A and VEGFR-2 mRNA and protein. The results suggest that EELC inhibits tumor angiogenesis through inhibiting the proliferation and migration of HUVECs, and by downregulating VEGF and VEGFR.

Pien Tze Huang Gan Bao attenuates carbon tetrachloride­induced hepatocyte apoptosis in rats, associated with suppression of p53 activation and oxidative stress.

Pien Tze Huang Gan Bao (PZH­GB), a traditional Chinese medicine, has been used for thousands of years as a protective remedy effective against liver injury induced by excessive alcohol and smoking. The present study aimed to evaluate the protective effects and potential mechanisms of PZH­GB against carbon tetrachloride (CCl4)­induced hepatic injury. Rats were pre­treated with silymarin (50 mg/kg) or different doses of PZH­GB (150, 300 or 600 mg/kg) orally administered for 7 days. At the end of treatment, the rats were intraperitoneally injected with CCl4, or control rats received a corn oil injection. The lactate dehydrogenase (LDH) levels in serum were evaluated. Apoptosis was assessed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. p53, B­cell lymphoma 2 (Bcl­2), B cell­lymphoma 2­associated X protein (Bax), cyclooxygenase­2 (COX­2), inducible nitric oxide synthase (iNOS) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) were measured by reverse transcription­quantitative polymerase chain reaction and western blotting. The activity of caspase­9 and caspase­3 were measured by a colorimetric assay. The results indicated that silymarin and PZH­GB prevented CCl4­induced serum LDH elevations, and CCl4 induced high levels of LDH. Compared with the CCl4 group, silymarin and PZH­GB treatment significantly decreased LDH levels. Histopathological results revealed that silymarin and PZH­GB ameliorated the CCl4­induced liver histological alterations. The TUNEL results showed that compared with the control group, CCl4 induced liver cell apoptosis, while silymarin and PZH­GB treatment inhibited apoptosis and the TUNEL­positive cells. The elevated expression of Bax, p53, iNOS, COX­2 and CYP2E1 were reduced by silymarin or PZH­GB pretreatment, whereas reduced Bcl­2 expression levels were increased. CCl4 increased the activity of caspase­9 and ­3 by 6.86­ and 7.42­fold, respectively; however, silymarin and PZH­GB ameliorated this effect. In conclusion, silymarin and PZH­GB treatment prevented the deleterious effects on liver functions by attenuation of oxidative stress, inflammation and mitochondrial apoptosis via the p53 signaling pathway.