Cinobufacini Inhibits Survival and Metastasis of Hepatocellular Carcinoma via c-Met Signaling Pathway.

OBJECTIVE: To investigate the anti-tumor effects of cinobufacini (CINO) on hepatocellular carcinoma (HCC) induced by des-gamma-carboxy-prothrombin (DCP) and to uncover the underlying mechanisms. METHODS: The inhibitory effect of CINO on HCC cell proliferation was evaluated using the cell counting kit-8 method, and the apoptosis rate was quantified using flow cytometry. Immunofluorescence and Western blot analyses were used to investigate the differential expression of proteins associated with cell growth, apoptosis, migration, and invasion pathways after CINO treatment. The therapeutic potential of CINO for HCC was confirmed, and the possibility of combining cinobufacini with c-Met inhibitor for the treatment of primary HCC was further validated by in vivo experiments. RESULTS: Under the induction of DCP, CINO inhibited the activity of HCC cells, induced apoptosis, and inhibited migration and invasion. Upon the induction of DCP, CINO regulated c-Met activation and the activation of the phosphatidylinositol-3 kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways. In a mouse model of HCC, CINO exhibited significant antitumor effects by inhibiting the phosphorylation of c-Met and the downstream PI3K/AKT and MEK/ERK pathways in tumor tissues. CONCLUSIONS: CINO inhibited HCC cell growth, promoted apoptosis, and suppressed HCC cell invasion and migration by targeting c-Met and PI3K/AKT and MEK/ERK signaling pathways under DCP induction.

Adenovirus-mediated P311 ameliorates renal fibrosis through inhibition of epithelial-mesenchymal transition via TGF-ß1-Smad-ILK pathway in unilateral ureteral obstruction rats.

Epithelial-mesenchymal transition (EMT) is a critical step and key factor during renal fibrosis. Preventing renal tubular EMT is important for delaying the progression of chronic kidney disease (CKD). P311, a highly conserved 8-kDa intracellular protein, has been indicated as an important factor in myofibroblast transformation and in the progression of fibrosis. However, the related studies on P311 on renal fibrosis are limited and the mechanisms of P311 in the progression of renal tubulointerstitial fibrosis remain largely unknown. In the present study, we examined the effect of P311 on transforming growth factor-ß1 (TGF-ß1)-mediated EMT in a rat model of unilateral ureteral occlusion (UUO) renal fibrosis. The recombinant adenovirus p311 (also called Ad-P311) was constructed and transferred it into UUO rats, the preventive effect and possible mechanism of P311 on TGF-ß1-mediated EMT were explored. The UUO model was established successfully and Ad-P311 was administered into UUO rats each week for 4 weeks, then the serum levels of Cr, blood urea nitrogen (BUN) and albumin (ALB) were evaluated. H&E staining and Masson staining were performed to observe the pathological changes of kidneys. Immunohistochemical staining and western blot analysis were used to examine the EMT markers [E-cadherin and α-smooth muscle actin (α-SMA)], and signal transducers (p-Smad2/3 and Smad7). Integrin linked kinase (ILK) as a keyintracellular mediator that controls TGF-ß1-mediated-EMT was also assayed by western blot analysis. The results showed that P311 could alleviate renal tubular damage and interstitial fibrosis improving Cr, BUN and ALB serum levels in UUO kidneys. Furthermore, P311 attenuated TGF-ß1-mediated EMT through Smad-ILK signaling pathway with an increase in α-SMA, pSmad2/3 and ILK expression, and a decrease in E-cadherin and Smad7 expression in UUO kidneys. In conclusion, P311 may be involved in the pathogenesis of renal fibrosis by blocking TGF-ß1-mediated EMT via TGF-ß1-Smad-ILK pathway in UUO kidneys. P311 may be a novel target for the control of renal fibrosis and the progression of CKD.

Cost-effectiveness analysis of neonatal hearing screening program in China: should universal screening be prioritized?

BACKGROUND: Neonatal hearing screening (NHS) has been routinely offered as a vital component of early childhood care in developed countries, whereas such a screening program is still at the pilot or preliminary stage as regards its nationwide implementation in developing countries. To provide significant evidence for health policy making in China, this study aims to determine the cost-effectiveness of NHS program implementation in case of eight provinces of China. METHODS: A cost-effectiveness model was conducted and all neonates annually born from 2007 to 2009 in eight provinces of China were simulated in this model. The model parameters were estimated from the established databases in the general hospitals or maternal and child health hospitals of these eight provinces, supplemented from the published literature. The model estimated changes in program implementation costs, disability-adjusted life years (DALYs), average cost-effectiveness ratio (ACER), and incremental cost-effectiveness ratio (ICER) for universal screening compared to targeted screening in eight provinces. RESULTS AND DISCUSSION: A multivariate sensitivity analysis was performed to determine uncertainty in health effect estimates and cost-effectiveness ratios using a probabilistic modeling technique. Targeted strategy trended to be cost-effective in Guangxi, Jiangxi, Henan, Guangdong, Zhejiang, Hebei, Shandong, and Beijing from the level of 9%, 9%, 8%, 4%, 3%, 7%, 5%, and 2%, respectively; while universal strategy trended to be cost-effective in those provinces from the level of 70%, 70%, 48%, 10%, 8%, 28%, 15%, 4%, respectively. This study showed although there was a huge disparity in the implementation of the NHS program in the surveyed provinces, both universal strategy and targeted strategy showed cost-effectiveness in those relatively developed provinces, while neither of the screening strategy showed cost-effectiveness in those relatively developing provinces. This study also showed that both strategies especially universal strategy achieve a good economic effect in the long term costs. CONCLUSIONS: Universal screening might be considered as the prioritized implementation goal especially in those relatively developed provinces of China as it provides the best health and economic effects, while targeted screening might be temporarily more realistic than universal screening in those relatively developing provinces of China.

Ningdong granule: a complementary and alternative therapy in the treatment of attention deficit/hyperactivity disorder.

BACKGROUND: Attention deficit/hyperactivity disorder (ADHD) is a common neurobehavioral and neuropsychiatric disorder in school-age children, and recent studies provide evidence implicating the metabolic abnormalities of dopamine (DA) for its pathophysiology. Methylphenidate, a kind of psychostimulant, is widely used in the treatment of ADHD, but some patients do not respond to it or cannot bear its side effects. As a traditional Chinese medicine preparation, Ningdong granule (NDG) has been used in the treatment of ADHD for several years in China. However, a systematical pharmacological study on its safety and mechanism still remains obscure. OBJECTIVE: This paper aims to evaluate the efficiency, safety, and possible mechanism of NDG on ADHD children compared to methylphenidate. METHODS: Seventy-two ADHD children were recruited to perform an 8-week, randomized, methylphenidate-controlled, doubled-blinded trial. The subjects were equally assigned to two groups receiving either NDG 5 mg/kg/day or methylphenidate 1 mg/kg/day for 8 weeks. The efficiency was assessed by the Teacher and Parent ADHD Rating Scales every 2 weeks for a total of 8 weeks. The side effects were recorded during the study. Blood, urine, and stool routine samples, liver and renal function test, and DA and homovanillic acid (HVA) concentration in sera were tested at the beginning and end of the trial. RESULTS: NDG ameliorated ADHD symptoms after an 8-week medication with fewer side effects compared to methylphenidate (P < 0.05). The result also showed NDG to be safe and tolerable for ADHD children as monitored by the blood, urine, and stool analysis and liver and renal function for 8 weeks (P < 0.05). Moreover, the level of HVA in sera increased in NDG-treated group (P < 0.05), while the content of DA had no significant change during the study. An analysis of Pearson correlation coefficients also showed that the increased content of HVA in sera was associated with the improved scores of Teacher and Parent ADHD Rating Scales. CONCLUSIONS: Compared to methylphenidate, NDG is effective and safe for ADHD children in the short term, increases the HVA concentration in sera to regulate DA metabolism, and promises to be an alternative medication, safely and effectively.

[Apoptosis-inducing effect of cinobufacini on human hepatoma cell line HepG2 and its mechanism of action].

To investigate the apoptosis-inducing effect of cinobufacini (Huachansu) on human hepatoma cell line HepG2 and its possible mechanism of action, HepG2 cells were treated with different concentrations of cinobufacini. Cell proliferation was measured by methylthiazolyl tetrazolium (MTT) assay. The morphological changes of apoptosis were observed by Hoechst 33258 staining. Cell cycle distribution and apoptotic rate were evaluated by flow cytometry (FCM). Quantitative real-time RT-PCR and Western blotting analysis were used to detect the mRNA and protein expressions of apoptosis related factors Bcl-2, Bax and p53. The results indicated that cinobufacini could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. Remarkable morphological changes of apoptosis including cytoplasmic and nuclear condensation and partition of cytoplasm were observed by Hoechst 33258 staining. According to FCM analysis, HepG2 cells were arrested in G2/M phase and the apoptotic rate increased with the increase of the concentration of cinobufacini. Both the mRNA and protein expressions of Bax and p53 were up-regulated while Bcl-2 expression down-regulated. Thus, cinobufacini could inhibit the proliferation and induce apoptosis of human hepatoma cell line HepG2. Furthermore, up regulation of Bax and p53 as well as down regulation of Bcl-2 expressions may be one of the important apoptotic inducing mechanisms.

Apoptosis-inducing activity of compounds screened and characterized from cinobufacini by bioassay-guided isolation.

Cinobufacini (huachansu), an aqueous extract from the skin of the toad Bufo bufo gargarizans Cantor, is a traditional Chinese medicinal preparation widely used in clinical cancer therapy in China. Here, we screened and identified active compounds of cinobufacini and investigated their apoptosis-inducing effect on HepG2 cells. Screening was performed using bioassay-guided isolation. The effects of different fractions on the proliferation of HepG2 cells were detected by the MTT assay. The extraction and isolation of active fractions were performed by chloroform extraction, silica column chromatography, preparative thin-layer chromatography and high-performance liquid chromatography. Nuclear magnetic resonance (NMR) imaging and electron ionization-mass spectrometry (EI-MS) were used to identify the structure of the active compounds. The extent of cell apoptosis was detected by Hoechst 33258 staining and flow cytometric analysis. Western blot analysis was used to detect the expression of the apoptosis-related proteins Bax and Bcl-2. Through bioassay-guided isolation, two compounds were isolated from cinobufacini. NMR and EI-MS data revealed these compounds to be resibufogenin and cinobufagin. Cinobufagin was determined to be the more efficient of the two in inhibiting the proliferation of HepG2 cells. Hoechst 33258 staining and flow cytometric analysis indicated that cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells in HepG2 cells. Western blot analysis showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression. In conclusion, we screened and identified two anti-proliferation compounds of cinibufacini, resibufogenin and cinobufagin. The most effective compound, cinobufagin, inhibited cell proliferation by inducing the apoptosis of HepG2 cells. This was potentially triggered by regulation of the Bax/ Bcl-2 ratio.