[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.

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.

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.

Pien Tze Huang Gan Bao ameliorates carbon tetrachloride-induced hepatic injury, oxidative stress and inflammation in rats.

Liver damage results from a variety of insults, including hepatitis and chemical toxicity from alcohol, drugs and other toxins. The present study evaluated the hepatoprotective effects and potential mechanisms of action of the Traditional Chinese Medicine Pien Tze Huang Gan Bao (GB) in a rat model of carbon tetrachloride (CCl4)-induced liver injury. Sixty male Sprague-Dawley rats were randomly divided into six different groups: i) Control, ii) CCl4 injury model and groups treated with iii) silymarin as a positive drug control, iv) 150 mg/kg GB, v) 300 mg/kg GB and vi) 600 mg/kg GB. Control rats received no treatment, while the remaining ones were intraperitoneally injected with CCl4 (2 ml/kg) to induce acute liver disease. Silymarin or GB was orally administered prior to CCl4 treatment in various treatment groups for 7 days. Animals were sacrificed 24 h post-CCl4 injection. It was revealed that GB significantly reduced serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase and total bilirubin levels in the serum induced by CCl4. BG also prevented CCl4-induced changes in liver tissues, as revealed by histopathological analysis. CCl4-induced reductions in endogenous liver antioxidant enzyme activities of superoxide dismutase, glutathione and glutathione peroxidase as well as increases in malondialdehyde and thiobarbituric acid reactive substances were inhibited by GB treatment. Activated NF-κB in liver tissues was also significantly increased by CCl4, which was attenuated by GB as indicated by immunohistochemical and PCR analysis. Furthermore, CCl4-mediated increases in the inflammatory factors tumor necrosis factor-alpha and interleukin-1ß secretion into the serum and their expression in liver tissues were reversed following GB treatment, as revealed by ELISA and PCR, respectively. These findings suggested that GB protects against CCl4-induced hepatic injury, inflammation and oxidative damage in rats and may be useful in future clinical application of liver injury and disease.

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.

Artemisia capillaris formula inhibits hepatic steatosis via an miR­122­induced decrease in fatty acid synthase expression in vivo and in vitro.

Non­alcoholic fatty liver disease (NAFLD) is a widespread health concern, and there is currently insufficient understanding regarding its pathogenesis and treatment. The present study aimed to explore the effects of Artemisia capillaris formula (ACF) on high­fat diet­induced hepatic steatosis and fatty acid­induced intracellular lipid accumulation, by micro (mi)RNA regulation. A total of 72 Sprague­Dawley rats were divided into six groups (n=12/group). One group was designated as the control group and fed a normal diet, and the remaining five groups were allowed ad libitum access to a high­fat diet for eight weeks, in order to establish an NAFLD rat model. The rats were subsequently administered polyene phosphatidylcholine (PP; 0.076 g/kg body weight/day), low dose of ACF (0.462 g/kg body weight/day), middle dose of ACF (0.924 g/kg body weight/day) or high dose of ACF (1.848 g/kg body weight/day) intragastrically for four weeks. HepG2 human hepatocellular carcinoma cells were treated with oleic acid and palm acid, followed by treatment with various concentrations of ACF. Serum alanine transaminase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), high­density lipoprotein cholesterol (HDL­C), low­density lipoprotein cholesterol (LDL­C), and steatotic HepG2 human liver carcinoma cell TC and TG levels were measured. ACF and PP treatments attenuated high­fat diet­induced hepatic steatosis and fatty acid­induced intracellular lipid accumulation. A modified high­fat diet significantly increased ALT, AST, TG, TC, LDL­C levels and decreased HDL­C levels. Treatment with ACF and PP abrogated the increase in liver enzymes and TG, TC and LDL­C levels, but did not influence HDL­C levels in a high­fat diet induced rat model of steotosis. Steatotic HepG2 cells exhibited significantly increased levels of both TG and TC. Treatment with ACF significantly decreased TC and TG levels in vivo, and ACF and PP treatment decreased the expression levels of fatty acid synthase (FASN) and increased miR­122 in vivo and in vitro. In conclusion, these results suggested that ACF may inhibit hepatic steatosis via miR­122­induced downregulation of FASN in vivo and in vitro.

Opposing needling promotes behavior recovery and exerts neuroprotection via the cAMP/PKA/CREB signal transduction pathway in transient MCAO rats.

The aim of the present study was to investigate whether the cyclic adenosine 3',5'­monophosphate (cAMP)/protein kinase A(PKA)/cAMP­responsive element binding protein (CREB) signal transduction pathway triggered by γ­aminobutyric acid class B (GABA(B)) receptor activation is involved in neuroprotection against ischemia and behavioral recovery induced by opposing needling (ON). A total of 80 rats were randomly divided into four groups: A sham operation group, an ischemia group, an ON group and an ON group effectively inhibited by the GABA(B) receptor antagonist, CGP35384 (n=20/group). The behavior of the rats was assessed by their neurological deficit score, whereas the impairment of gait was examined using the CatWalk system. The volume of cerebral infarction was examined upon treatment with 2,3,5­triphenyltetrazolium chloride. The expression levels of CREB, GABA(B1) and GABA(B2) were examined by western blotting and reverse transcription­quantitative polymerase chain reaction, and the activity of adenylyl cyclase (AC), cAMP and PKA in the serum was detected using an enzyme­linked immunosorbent assay. In the present study, in comparison with other groups, the ON group exhibited a reduced score for the neurological deficit, the stride length and swing speed were improved, and the volume of infarction was reduced. However, these effects were reversed upon administration of CGP35384. Additionally, the expression levels of CREB, GABA(B1) and GABA(B2) were increased in the ON group. The levels of AC, cAMP and PKA in the serum were also increased in the ON group, whereas the addition of CGP35384 reversed these effects. The results of the present study demonstrated that ON markedly protected the brain against transient cerebral ischemic injury, and this effect was possibly mediated by the activation of the GABAB/cAMP/PKA/CREB signal transduction pathway. These findings implied that ON may be a potential therapeutic method for treating stroke.

miRNA Regulation Network Analysis in Qianliening Capsule Treatment of Benign Prostatic Hyperplasia.

Objective. The objective of this study was to evaluate the molecular mechanism by which Qianliening capsule (QC) treats benign prostatic hyperplasia (BPH). Methods. Benign prostatic hyperplasia epithelial cell line BPH-1 was treated with 0, 1.25, 2.5, and 5 mg/mL QC for 48 h, respectively. Evaluation of cell viability and observation of morphologic changes of BPH-1 cell gene expression and miRNA expression profiles were analyzed. Real-time quantitative PCR was used to confirm changes in miRNA and gene expression. GO and KEGG pathway-based approaches were used to investigate biological functions and signaling pathways affected by differentially expressed mRNAs. Results. QC inhibited BPH-1 cell proliferation. Differential expression of 19 upregulated and 2 downregulated miRNAs was observed in QC-treated BPH-1 cells compared to untreated control cells. 107 upregulated and 71 downregulated genes were identified between the two groups. Significantly enriched signaling pathways based on deregulated mRNAs were mainly involved in regulation of cell proliferation, apoptosis, and so on. Additionally, miRNA-mRNA network analysis integrated these miRNAs and genes by outlining interactions of miRNA and related genes. Conclusion. The study was the first report of differentially expressed miRNA and mRNA in QC-treated BPH-1 cells.

Inhibition of Hepatocellular Carcinoma by Total Alkaloids of Rubus alceifolius Poir Involves Suppression of Hedgehog Signaling.

OBJECTIVE: We evaluated the effects of total alkaloids of Rubus alceifolius Poir (TARAP) on the migration and invasion of hepatocellular carcinoma (HCC) and furthermore investigated the possible molecular mechanisms mediating its anticancer activity. METHODS: We implanted nude mice with human HCC HepG2 cells and fed them with vehicle (physiological saline) or 3 g/kg/day dose of TARAP 5 days per week for 21 days. We determined the in vitro effect of TARAP on the migration and invasion of HepG2 cells by transwell assay. We evaluated SHH signaling components' (SHH, PTCH, SMO, and Gli1) expression levels by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Activity of the matrix metalloproteinases (MMPs) in supernatants was analyzed by zymography. The expression of the MMPs and their specific tissue inhibitor (tissue inhibitor of matrix metalloproteinases, TIMP-1, 2) in HCC tissues was detected by immunohistochemistry. RESULTS: We discovered that TARAP inhibited hepatocellular migration and invasion in a dose-dependent manner in vitro. In addition, TARAP decreased the expression of SHH, PTCH, SMO, and Gli1 in HCC mouse tumors at both transcriptional and translational levels. Moreover, TARAP inhibited the activity of MMP2 and MMP9. We found that TARAP reduced the expression of MMP2 and MMP9, as well as the tissue inhibitor of MMPs. CONCLUSION: Our study showed that TARAP inhibits HCC migration and invasion likely through suppression of the hedgehog pathway. This may, in part, explain its anticancer properties. These results suggest that total alkaloids in Rubus alceifolius may have potential as a novel antimetastasis drug in the treatment of HCC.

Anti-proliferative effects of qianliening capsules on prostatic hyperplasia in vitro and in vivo.

Previous studies by our group showed that Qianliening capsules (QC), a clinically proven effective traditional Chinese formulation that has long been used in the treatment of benign prostatic hyperplasia (BPH), is capable of inhibiting BPH in vivo and in vitro via the promotion of apoptosis, suppression of the EGFR/STAT3 signaling pathway and regulating the expression of sex hormones as well as their receptors. However, the mechanism of its anti-BPH activity has remained to be fully elucidated. The present study aimed to investigate the mechanism underlying the anti-proliferative effect of QC in vivo and in vitro. Castrated male Sprage-Dawley (SD) rats where subcutaneously injected with testosterone propionate and the WPMY-1 cell line was stimulated with basic fibroblast growth factor in order to generate BPH in vivo and in vitro separately, both of which were then subjected to QC treatment. Finasteride was used as a positive control drug for the in vivo study. In the present study, it was found that treatment with QC or finasteride significantly reduced the prostatic index (PI=prostate wet weight/body weight x 100) in a rat model of BPH (P<0.05). In addition, reverse transcription quantitative polymerase chain reaction (RT-PCR) and western blot analyses showed that QC or finasteride treatment significantly inhibited model construction-induced upregulation of expression of proliferating cell nuclear antigen, cyclin D1 and cyclin-dependent kinase 4 in prostatic tissues of rats with BPH (P<0.05). The in vitro study further proved that QC exhibited anti-proliferative properties via G1/S cell cycle arrest in the WPMY-1 cell line, as evidenced by colony formation, flow cytometric cell cycle, immunoblot and RT-PCR analyses. In conclusion, the present study demonstrated that inhibition of cell proliferation via G1/S cell cycle arrest may be one of the underlying mechanisms of the effect of QC on BPH.

Bear bile powder inhibits angiogenesis in vivo and in vitro.

OBJECTIVE: To evaluate the effect of bear bile powder (BBP) on angiogenesis, and investigate the underlying molecular mechanisms. METHODS: A chick embryo chorioallantoic membrane (CAM) assay was used to evaluate the angiogensis in vivo. Human umbilical vein endothelial cells (HUVECs) were treated with 0, 0.25, 0.5, 0.75, and 1.0 mg/mL of BBP for 24, 48 and 72 h, respectively. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the viability of HUVECs. Cell cycle progression of HUVECs was examined by fluorescence-activated cell sorting (FACS) analysis with propidium iodide staining. HUVEC migration was determined by wound healing method. An ECMatrix gel system was used to evaluate the tube formation of HUVECs. The mRNA and protein expression of vascular endothelial growth factor (VEGF)-A in both HUVECs and HepG2 human cells were examined by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively. RESULTS: Compared with the untreated group, BBP inhibited angiogenesis in vivo in the CAM model (P< 0.01). In addition, treatment with 0.25-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability by 14%-27%, 29%-69% and 33%-91%, compared with the untreated control cells (P< 0.01). Additionally, BBP inhibited the proliferation of HUVECs via blocking the cell cycle G to S progression, compared with the S phase of untreated cells 48.05%± 5.00%, 0.25-0.75 mg/mL BBP reduced S phase to 40.38%± 5.30%, 36.54± 4.50% and 32.13± 3.50%, respectively (Pglt; 0.05). Moreover, BBP inhibited the migration and tube formation of HUVECs, compared with the tube length of untreated cells 100%± 12%, 0.25-0.75 mg/mL BBP reduced the tube length to 62%± 9%, 43%± 5% and 17%± 3%, respectively (p< 0.01). Furthermore, BBP treatment down-regulated the mRNA and protein expression levels of VEGF-A in both HepG2 cells and HUVECs. CONCLUSION: BBP could inhibit the angiogenesis by reducing VEGF-A expression, which may, in part, explain its anti-tumor activity.

Qianliening capsules influence the apoptosis of benign prostatic hyperplasia epithelial-1 cells by regulating the extracellular matrix.

The present study investigated whether Qianliening capsules (QC) affected the apoptosis of benign prostatic hyperplastia epithelial (BPH­1) cells by regulating the extracellular matrix (ECM). The levels of fibronectin (FN) and collagen IV were determined in the culture medium of BPH­1 cells maintained in normal medium and of BPH­1 cells maintained in an environment rich in FN and collagen IV using an enzyme­linked immunosorbent assay. Reverse transcription quantitative polymerase chain reaction and western blot analysis were performed to determine the mRNA and protein expression levels of FN, collagen IV, B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax) and cyclin D1, respectively. The cell morphology and viability were determined using light microscopy and an MTT assay and cell apoptosis was detected by annexin V staining. The results demonstrated that FN and collagen IV affected the apoptotic response of the BPH­1 cells, QC treatment significantly reduced the levels of FN and collagen IV secreted by the cells into the culture medium (P<0.01), inhibited the mRNA and protein expression levels of FN, collagen IV, Bcl­2 and cyclin D1 and promoted the mRNA and protein expression of Bax. Therefore, one of the mechanisms underlying the anti­BPH action of QC involves promoting apoptosis by regulating the expression of the extracellular matrix.

Inhibition of the signal transducer and activator of transcription 3 signaling pathway by Qianliening capsules suppresses the growth and induces the apoptosis of human prostate cells.

The signal transducer and activator of transcription 3 (STAT3) pathway is one of the main growth factor­mediated signal transduction pathways and is closely associated with the occurrence and development of benign prostatic hyperplasia (BPH). Qianliening capsules (QC) have significant therapeutic effects on BPH; however, the precise mechanism underlying its anti­BPH activity remains to be elucidated. To further elucidate the molecular mechanism of the therapeutic effect of QC on BPH, the present study used epidermal growth factor (EGF), which has a role in the pathogenesis of BPH, to stimulate the growth of human prostate WPMY­1 cells and activate the STAT3 pathway in the WPMY­1 cells. The cell viability was determined using an MTT assay and the cell morphology was observed by phase­contrast microscopy. Fluorescence activated cell sorting analysis with Annexin­V/propidium iodide (PI) staining and PI staining were performed to examine cell apoptosis and the cell cycle. The activation of caspase­9 and ­3 were evaluated by colorimetric assay. STAT3 phosphorylation and transcriptional activity were detected by western blot analysis and the luciferase gene reporter, respectively. The mRNA and protein expression levels of B­cell lymhoma 2 (Bcl­2), Bcl­2­associated X protein (Bax), cyclin D1, cyclin­dependent kinase 4 (CDK4) and p21 were measured by reverse transcription quantitative polymerase chain reaction and western blot analysis, respectively. In the present study, QC was found to significantly and dose­dependently inhibit the EGF­stimulated growth of WPMY­1 cells, as evidenced by QC­induced cell -morphological changes and a reduction in cell viability. In addition, QC treatment markedly induced the activation of caspase­9 and ­3. QC treatment also inhibited the EGF­mediated increase of STAT3 phosphorylation levels and transcriptional activity in WPMY­19 cells, accompanied by downregulation of the expression of Bcl­2, cyclin D1 and CDK4 and upregulation of the expression of Bax and p21. These results suggested that QC effectively inhibited the proliferation and promoted the apoptosis of human prostate cells via modulation of the STAT3 signaling pathway and its target genes, which is likely to be one of the mechanisms underlying its activity in BPH treatment.

Treatment of Nonalcoholic Fatty Liver Disease with Total Alkaloids in Rubus aleaefolius Poir through Regulation of Fat Metabolism.

Total alkaloids in Rubus aleaefolius Poir (TARAP) is a folk medicinal herb that has been used clinically in China to treat nonalcoholic fatty liver disease (NAFLD) for many years. However, the mechanism of its anti-NAFLD effect is largely unknown. In this study, we developed a NAFLD rat model by supplying a modified high-fat diet (mHFD) ad libitum for 8 weeks and evaluated the therapeutic effect of TARAP in NAFLD rats as well as the underlying molecular mechanism. We found that TARAP could reduce the serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL-C) levels and increase the serum high-density lipoprotein (HDL-C) level in NAFLD rats. In addition, TARAP treatment reduced expression of fatty acid synthetase (FAS), and acetyl-CoA carboxylase (ACC) and upregulated the expression of carnitine palmitoyltransferase (CPT). Our results suggest that regulation of lipid metabolism may be a mechanism by which TARAP treats NAFLD.

Clinical study on Kangquan Recipe (康泉方) for benign prostatic hyperplasia patients: a randomized controlled trial.

OBJECTIVE: To observe the effectiveness and safety of Kangquan Recipe (康泉方, KQR) for benign prostatic hyperplasia (BPH) patients. METHODS: One hundred and six BPH patients were randomly assigned to the treatment group (53 cases) and the control group (53 cases) according to a random number table. The treatment group was given KQR orally; the control group was given cernilton orally. After 24-week treatment, the clinical effect and safety were evaluated using the International Prostatic Symptom Score (I-PSS), quality of life (QOL), maximum flow rate (Qmax), average flow rate (Qave), residual urine volume (RUV), total prostatic volume (TPV), etc. RESULTS: After treatment, the score of I-PSS was decreased from 16.9±5.6 to 12.5±4.6 in the treatment group, significantly lower compared with the control group; the levels of Qmax and Qave were from 10.9±3.5 to 15.6±4.5 and 5.4±2.1 to 7.3±2.5 (mL/s) in the treatment group, significantly higher compared with the control group; the levels of RUV and TPV were from 70.8±28.2 to 35.2±21.8 and 37.2±16.9 to 30.1±10.8 (mL) in the treatment group, significantly lower compared with the control group (all P<0.05). The incidence rate of adverse reaction was similar between the two groups (P>0.05). CONCLUSION: KQR is effective and safe for the treatment of BPH.

Treatment of benign prostatic hyperplasia with Croton membranaceus in an experimental animal model.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton membranaceus leaf extracts are used in the Bahamas to aromatize tobacco. In Nigeria it is used to improve digestion and in Ghana, the root extract is used for the treatment of benign prostatic hyperplasia (BPH). Despite claims of efficacy no data exists to support this. The aim of this study was to determine if Croton membranaceus aqueous root extract (CMARE) could attenuate the development of BPH in an animal model. MATERIALS AND METHODS: Fifty (50) adult male Sprague-Dawley rats weighing 200-250g were randomly divided into 5 groups. Group 1 served as the control and received normal saline p.o. Groups 2-5 were castrated and injected with 5mg/kg b.wt. testosterone propionate subcutaneously for 28 days. Group 2 (model group) had no further treatment. Group 3 was simultaneously given 0.5mg/kg b.wt. finasteride p.o. throughout. Groups 4 and 5 received 30mg/kg b.wt. [low dose (LD)] and 300mg/kg b.wt. [high dose (HD)] CMARE, respectively, for 28 days. Rats were sacrificed at the end of the study and all prostate organs harvested. Wet weights, volumes and prostatic index (PI) were determined. Tissues were histologically examined. Serum prostate specific antigen (PSA) and dihydrotestosterone (DHT) levels were determined. RESULTS: Prostate volume of the control group was 0.67±0.23cm(3). The model, finasteride, CMARE LD and HD groups had the following volumes: 0.92±0.12, 0.84±0.16, 0.79±0.16 and 0.80±0.19cm(3), respectively. Only the model group showed significant statistical differences with the control (p=0.007). PI for control, model, finasteride, LD and HD groups was as follows: 0.19±0.04, 0.30±0.04, 0.25±0.04, 0.21±0.05 and 0.22±0.05. No statistical differences between the control PI and the CMARE treated groups were observed. Histologically, the model group had massive growth of columnar stromal and epithelial cells. CMARE and finasteride attenuated this growth with a resultant thin layer of stromal and epithelial cells similar to the control. PSA levels were significantly lower in the treatment groups. CONCLUSION: CMARE reduces stromal and epithelial cell growth, and subsequently shrinks enlarged prostate. This is the first scientific proof validating the anecdotal evidence of CMARE efficacy in the management of BPH.

[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.

Total alkaloids of Rubus alceifolius Poir shows anti-angiogenic activity in vivo and in vitro.

Total alkaloids is an active ingredient of the natural plant Rubus alceifolius Poir, commonly used for the treatment of various cancers. Antitumor effects may be mediated through anti-angiogenic mechanisms. As such, the goal of the present study was to investigate and evaluate the effect of total alkaloids in Rubus alceifolius Poir (TARAP) on tumor angiogenesis and investigate the underlying molecular mechanisms of TARAP action in vivo and in vitro. A chick embryo chorioallantoic membrane (CAM) assay was used to assess angiogenesis in vivo. An MTT assay was performed to determine the viability of human umbilical vein endothelial cells (HUVECs) with and without treatment. Cell cycle progression of HUVECs was examined by FACS analysis with propidium iodide staining. HUVEC migration was determined using a scratch wound method. Tube formation of HUVECs was assessed with an ECMatrix gel system, and mRNA and protein expression of VEGF-A in both HUVECs and HepG2 human hepatocellular carcinoma cells were examined by RT-PCR and ELISA, respectively. Our results showed that TARAP inhibited angiogenesis in the CAM model in vivo and inhibited HUVEC proliferation via blocking cell cycle G1 to S progression in a dose- and time-dependent manners in vitro. Moreover, TARAP inhibited HUVEC migration and tube formation and downregulated mRNA and protein expression of VEGF-A in both HepG2 cells and HUVECs. Our findings suggest that the anti-angiogenic activity of TARAP may partly contribute to its antitumor properties and may be valuable for the treatment of diseases involving pathologic angiogenesis such as cancer.

Protective effects and mechanisms of total alkaloids of Rubus alceaefolius Poir on non­alcoholic fatty liver disease in rats.

The plant Rubus alceaefolius Poir is used as a hepatic protectant in Traditional Chinese Medicine. The aim of the present study was to confirm the protective effect of the total alkaloids of Rubus alceaefolius Poir (TARAP) on the liver and to evaluate the potential molecular mechanisms associated with adipocytokines underlying non-alcoholic fatty liver disease (NAFLD) in rats. To generate the NAFLD model, Sprague-Dawley rats were administered a high­fat diet and following 12 weeks of model construction, rats were orally treated with a positive control drug and different doses of TARAP daily for 28 days. The rats were then sacrificed and the livers were collected to evaluate the liver index (LI) and observe histological changes by hematoxylin and eosin (H&E) staining. The secretion levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were examined by ELISA. Finally, the expression levels of leptin (LEP), resistin and adiponectin (APN) in liver tissues were determined by immunohistochemistry (IHC). The results demonstrated that, in the group treated with methionine and choline bitartrate tablets and in the groups treated with different doses of TARAP, there was a significant reduction in the LI (P<0.05 or P<0.01), a downregulation of the secretion levels of ALT and AST, reduced levels of LEP and resistin and an increased expression of APN in the liver of NAFLD rats compared with the model group. Furthermore, the effect of TARAP treatment of NAFLD rats was dose dependent. In conclusion, TARAP is a potential agent for downregulating LEP and resistin and upregulating APN expression in rats with NAFLD. Furthermore, TARAP may be a potential candidate for improving treatment responses in patients with NAFLD.