Pharmacokinetics of Nitazoxanide Dry Suspensions After Single Oral Doses in Healthy Subjects: Food Effects Evaluation and Bioequivalence Study.

Nitazoxanide (NTZ) is an effective antiparasitic drug with potent antiviral and antimicrobial activity. This randomized, open-label, 2-sequence, 2-period crossover trial was designed to evaluate the bioequivalence (BE) of the NTZ dry suspension in healthy subjects and investigated the effect of food intake on the pharmacokinetic (PK) properties of tizoxanide (an active metabolite of NTZ, TIZ). Sixty healthy Chinese subjects were enrolled and received a single dose of 500 mg/25 mL of preparations on days 1 and 4 under overnight fasting or fed conditions, respectively. The plasma concentration of TIZ was determined using high-performance liquid chromatography/tandem mass spectrometry. PK parameters were calculated using WinNonlin 8.2 and BE was evaluated using SAS 9.4. The 90% confidence intervals for the geometric mean ratio (test/reference) of maximum concentration (Cmax), the area under the curve from time 0 to the time of the last quantifiable concentration (AUC0-t), and the area under the curve from time 0 to extrapolation to infinity (AUC0-∞) were all within the equivalent interval of 80%-125%, compliant with BE requirements. In comparison with fasting, on taking the reference and test preparations of the NTZ dry suspension after a meal, the AUC0-t increased by 48.9% and 47.3%, respectively, the AUC0-∞ increased by 48.4% and 48.3%, respectively, and the post-meal Tmax was prolonged by 1.8-2 hours. Our results demonstrate that the test and reference preparations were bioequivalent. High-fat meals significantly improve the degree of drug absorption and delay the rate of drug absorption.

[Research Progress on Vector Flow Imaging of Cardiac Ultrasound].

Echocardiogram is vital for the diagnosis of cardiac disease. The heart has complex hemodynamics requiring an advanced ultrasound imaging mode. Cardiac ultrasound vector flow imaging is capable of measuring the actual magnitude and direction of the blood flow velocity, obtaining the quantitative parameters of hemodynamics, and then providing more information for clinical research and diagnosis. This study mainly reviewed several different vector flow imaging techniques for cardiac flow and presented the implementation difficulties, and proposed a diverging wave based high frame rate cardiac ultrasound vector flow imaging. The study discussed the limitation of current ultrasound technology used in the cardiac flow measurement, analyzed and demonstrated the specific reasons for these implementation difficulties and the potential future development.

Chemical constituents of Viscum coloratum (Kom.) Nakai and their cytotoxic activities.

A new diarylheptanoid, (1 R,2S,3S,5S)-2,3-dihydroxy-3',3''-dimethoxy-4'-de-O-methylcentrolobine (1) and a new bisabolane-type sesquiterpenoid, (1 R,7S)-1,12,13-trihydroxybisabola-3,10-diene (2), together with nineteen known compounds (3-21) were isolated from the EtOH extract of the stems and branches of Viscum coloratum (Kom.) Nakai. Their structures were elucidated by extensive analysis of 1 D and 2 D NMR spectra and from the HRESIMS. All the compounds were evaluated for their cytotoxic activity against eight human tumor cell lines.

Oxidative stress promotes ventilator-induced lung injury through activating NLRP3 inflammasome and TRPM2 channel.

Ventilator has been widely used for life support, but ventilator-induced lung injury (VILI) is still a major problem. Oxidative stress has been considered as a key contributor for VILI, but the specific mechanism remains unclear. The expression of NLRP3 inflammasome in cells and inflammatory factors in the supernatant were measured. Mitochondrial ROS and TRPM2 channel currents were investigated using flow cytometry and Patch-clamp technique, respectively. TRPM2-/- and NLRP3-/- mice were used for animal experiments. Lung tissues were stained by HE and the wet-dry ratio, bronchoalveolar lavage fluid (BALF) protein, MPO (marrow peroxidase), NLRP3 inflammasome were also investigated. Knockdown of NLRP3 or Caspase-1 or treatments with SS-31 or YVAD inhibited the expression of the NLRP3 inflammasome, and reduced IL-1ß and IL-18 levels in cell supernatant. These treatments suppressed the production of ROS and lowered the TRPM2 channel currents, but Rotenone exerted an opposite effect. High-tidal volume ventilation significantly increased the levels of IL-1ß, IL-18, NLRP3 inflammasome, wet-dry ratio of lung, MPO and BALF protein. However, these parameters were down-regulated in TRPM2-/- and NLRP3-/- mice. These parameters were suppressed in TRPM2-/- and NLRP3-/- mice indicate that oxidative stress might promote VILI through activating NLRP3 inflammasome and TRPM2 channel.

Salidroside prevents hydroperoxide-induced oxidative stress and apoptosis in retinal pigment epithelium cells.

Salidroside (SAL) is the major pharmacologically active constituent of Rhodiola rosea, which possesses a wide range of pharmacological functions, including anti-aging, anti-inflammatory, antioxidant, anticancer and neuroprotective activities. However, the effects and mechanisms of SAL on oxidative stress in retinal pigment epithelial (RPE) cells exposed to hydrogen peroxide (H2O2) remain unclear. The present study investigated the protective effects of SAL and the underlying mechanisms against H2O2-induced oxidative stress in human RPE cells. ARPE-19 cells were treated with various doses of SAL for 24 h and then exposed to 200 µM H2O2 for 24 h. Cell viability was analyzed by a MTT assay, and the intracellular levels of reactive oxygen species were measured using CellROX orange reagent. Cell apoptosis was analyzed by annexin V/propidium iodide double staining, followed by flow cytometry. The levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein, phospho (p)-protein kinase B (Akt), Akt, p-glycogen synthase kinase (GSK)-3ß and GSK-3ß were evaluated using western blotting. The results demonstrated that SAL markedly attenuated H2O2-induced loss of cell viability. SAL also ameliorated H2O2-induced oxidative stress and cell apoptosis in RPE cells. In addition, pretreatment with SAL significantly increased the phosphorylation levels of Akt and GSK-3ß in H2O2-treated ARPE-19 cells. In conclusion, the present study demonstrated that SAL protected RPE cells against H2O2-induced cell injury through the activation of the Akt/GSK-3ß signaling pathway. This suggests that SAL may be a potential therapeutic strategy for the treatment of age-related macular degeneration.

Dexmedetomidine reduces ventilator-induced lung injury (VILI) by inhibiting Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling pathway.

Mechanical ventilation (MV) may lead to ventilator-induced lung injury (VILI). Previous research has shown that dexmedetomidine attenuates pulmonary inflammation caused by MV, but the underlying mechanisms remain unclear. Our study aims to test whether dexmedetomidine has a protective effect against VILI and to explore the possible molecular mechanisms using the rat model. Thirty adult male Wistar rats weighing 200-250 g were randomly assigned to 5 groups (n = 6): control, low tidal volume MV (LMV), high tidal volume (HVT) MV (HMV), HVT MV + dexmedetomidine (DEX), HVT MV + dexmedetomidine + yohimbine (DEX+Y). Rats were euthanized after being ventilated for 4 hours. Pathological changes, lung wet/dry (W/D) weight ratio, lung myeloperoxidase (MPO) activity, levels of inflammatory cytokines (i.e., interleukin [IL]-1ß, tumor necrosis factor alpha [TNF-α], and IL-6) in the bronchoalveolar lavage fluid (BALF) and lung tissues, expression of Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB, and activation of NF-κB in lung tissues were measured. Compared with HMV, DEX group showed fewer pathological changes, lower W/D ratios and decreased MPO activity of the lung tissues and lower concentrations of the inflammatory cytokines in the BALF and lung tissues. Dexmedetomidine significantly inhibited the expression of TLR4 and NF-κB and activation of NF-κB. Yohimbine partly alleviated the effects of dexmedetomidine. Dexmedetomidine reduced the inflammatory response to HVT-MV and had a protective effect against VILI, with the inhibition of the TLR4/NF-κB signaling pathway, at least partly via α2-adrenoceptors.

The protective effect of dopamine on ventilator-induced lung injury via the inhibition of NLRP3 inflammasome.

Dopamine (DA), a neurotransmitter, was previously shown to have anti-inflammatory effects. However, its role in ventilator-induced lung injury (VILI) has not been explicitly demonstrated. This study aimed to investigate the therapeutic efficacy and molecular mechanisms of dopamine in VILI. Rats were treated with dopamine during mechanical ventilation. Afterwards, the influence of dopamine on histological changes, pulmonary edema, the lung wet/dry (W/D) ratio, myeloperoxidase (MPO) activity, polymorphonuclear(PMN)counts, inflammatory cytokine levels, and NLRP3 inflammasome protein expression were examined. Our results showed that dopamine significantly attenuated lung tissue injury, the lung W/D ratio, MPO activity and neutrophil infiltration. Moreover, it inhibited inflammatory cytokine levels in the Bronchoalveolar lavage fluid (BAL). In addition, dopamine significantly inhibited ventilation-induced NLRP3 activation. Our experimental findings demonstrate that dopamine exerted protective effects in VILI by alleviating the inflammatory response through inhibition of NLRP3 signaling pathways. The present study indicated that dopamine could be a potential effective therapeutic strategy for the treatment of VILI.

Upregulation of Leucine Zipper Protein mRNA in Hepatocellular Carcinoma Associated With Poor Prognosis.

BACKGROUND: Leucine zipper protein (LUZP) plays key roles in development. Overexpression of LUZP was documented in several types of solid tumors. In this study, expression of LUZP messenger RNA (LUZP mRNA) in human hepatocellular carcinoma (HCC) was examined, and the correlations of LUZP mRNA level with patients' characteristics and prognosis were also investigated. METHODS: Total RNA was extracted from HCC and paired noncancerous liver tissues of 77 patients. Expression of LUZP mRNA in the tissues was determined by real-time quantitative reverse transcriptase polymerase chain reaction. Using average LUZP mRNA level in noncancerous liver tissues as the cutoff, patients with HCC were categorized into high-expression group and low-expression group. Correlations of LUZP mRNA with clinical parameters were analyzed. Overall survival of the patients in the 2 groups was analyzed by Kaplan-Meier method. RESULTS: The LUZP mRNA level was significantly higher in HCC samples than in the noncancerous liver tissues (1.87 ± 0.11 vs 0.58 ± 0.05, P < .01). Significant differences were found between the 2 groups in terms of portal vein invasion, Tumor Lymph Node Metastasis (TNM) stage, and recurrence of HCC. The current study failed to find significant differences between the 2 groups in clinical characteristics such as age, gender, lymph node metastasis, hepatitis B virus infection, family HCC history, and alcohol intake. Overall survival in high-expression group was 12 months while that in the low-expression group was 34 months (P = .03). CONCLUSION: The LUZP mRNA is a prognostic indicator in HCC, and overexpression is associated with poor prognosis in patients with HCC.

Notch-1 signaling regulates astrocytic proliferation and activation after hypoxia exposure.

Activation of astrocyte has been implicated in the neonatal hypoxic brain injury. However, the mechanisms that control astrocytic activation and astrogliosis formation remain unknown. Here, we found that Notch-1 is upregulated and activated by hypoxia in astrocytes as confirmed by an increase in NICD and Hes-1 expression. Remarkably, blockade of Notch-1 signaling with the specific inhibitor DAPT suppressed astrocytic proliferation. In addition, both expression and secretion of inflammatory factor IL-1ß was inhibited in DAPT-pretreated astrocytes, while no significant change in TNF-α expression was detected. Most interestingly, GFAP and VEGF expression was suppressed by DAPT pretreatment in hypoxic astrocytes and further confirmed in neonatal rats following hypoxic brain injury. Furthermore, inhibition of Notch caused a remarkable decrease in NF-κB/p65 expression and translocation. Moreover, downregulation of either VEGF or NF-κB/p65 reduced astrocytic proliferation. Taken together, these results demonstrated that Notch-1 signaling was activated in hypoxic astrocytes and regulated astrocytic proliferation and activation by suppressing VEGF or NF-κB/p65 signaling pathway. Therefore, Notch signaling is a potential therapeutic strategy in hypoxia brain damage.

Feasibility and safety of sorafenib treatment in hepatocellular carcinoma patients with spontaneous rupture.

AIM: To report the outcome of patients with ruptured hepatocellular carcinoma (HCC) treated at a single center during a 5-year period. METHODS: We retrospectively analyzed 32 patients who presented with ruptured HCC at Shandong Provincial Hospital Affiliated to Shandong University between 2008 and 2013. RESULTS: The mean age of the patients was 53 years (range 39-71 years). Of these patients, 22 received surgical management, 10 underwent transarterial embolization (TAE) or transarterial chemoembolization (TACE), and 12 received sorafenib after surgery, TAE or TACE. Cumulative survival rates at 4, 8 and 12 mo were 72.9%, 50.0% and 33.3%, respectively, in the surgery only group and were 90.0%, 80.6% and 64.1%, respectively, in the surgery plus sorafenib group. Cumulative survival rates at 4, 8 and 12 mo were 68.4%, 43.6% and 19.4%, respectively, in the surgery only or TAE/TACE only groups, and were 91.7%, 75.0% and 60.2%, respectively, in the sorafenib combination groups (P = 0.04). No unexpected side effects due to sorafenib were observed. The most common side effect was hand-foot skin reaction. To date, 5 patients have died. Median follow-up from the start of sorafenib therapy for the remaining 7 patients is 12.7 mo (range 5.8-32.2 mo). CONCLUSION: Sorafenib can be used in patients with ruptured HCC as it has interesting activity and is well tolerated; dose adjustment is generally not required. However, a larger prospective study is necessary to determine the efficacy of sorafenib in this group of patients.

Possible benefit of splenectomy in liver transplantation for autoimmune hepatitis.

Liver transplantation for autoimmune hepatitis (AIH) is usually successful with excellent long-term outcomes, but primary disease may recur. The recurrence of AIH is a significant cause of graft loss. This study was to analyze the effect of splenectomy in preventing AIH relapse. The clinical courses of 12 patients who had transplantation for AIH were analyzed retrospectively. All patients were subjected to transplantation for end-stage liver disease caused by chronic AIH. Based on the duration of immunosuppressive treatment before liver transplantation, simultaneous splenectomy was performed in ten patients. Two patients underwent liver transplantation without splenectomy, one of them developed recurrent AIH and died from graft failure caused by AIH relapse. However, no episode of AIH recurrence was observed in patients who had undergone simultaneous splenectomy. Splenectomy might be an option to prevent AIH relapse in some patients with high risk factors.

FH535 inhibits the proliferation of HepG2 cells via downregulation of the Wnt/ß-catenin signaling pathway.

Hepatocellular carcinoma (HCC) is a primary cancer of the liver. Target therapy may improve prognosis of HCC. In the present study, we evaluated the inhibition of the Wnt/ß-catenin pathway as a potential therapeutic approach. HepG2 cells were treated with the ß-catenin inhibitor FH535. ß-catenin protein expression was semi-quantitatively assessed using western blot analysis. Cell proliferation was examined with a 3­(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. The mRNA expression of nitric oxide synthase (iNOS) was detected by reverse transcription polymerase chain reaction. The Griess assay was used to determine nitric oxide (NO) concentration. FH535 inhibited the proliferation of HepG2 cells and decreased ß-catenin protein expression. mRNA expression of iNOS, a target gene of the Wnt/ß-catenin pathway, was decreased in FH535­treated HepG2 cells compared to the control group. NO production was also reduced by FH535. In conclusion, the ß-catenin inhibitor FH535 may inhibit HCC cell proliferation via downregulation of the Wnt/ß-catenin pathway. Thus, targeting this pathway may be useful in HCC therapy.

Construction of a recombinant eukaryotic human ZHX1 gene expression plasmid and the role of ZHX1 in hepatocellular carcinoma.

The zinc-fingers and homeoboxes protein 1 (ZHX1) consists of 873 amino acid residues, is localized in the cell nucleus and appears to act as a transcriptional repressor. Previous studies have shown that ZHX1 interacts with nuclear factor Y subunit α (NF-YA), DNA methyltransferases (DNMT) 3B and ZHX2, all of which are involved in tumorigenesis. However, the exact role of ZHX1 in tumorigenesis remains unknown. The aim of the current study was to construct a recombinant eukaryotic expression plasmid containing the human ZHX1 (hZHX1) gene and to investigate the biological activities of ZHX1 in hepatocellular carcinoma (HCC). Reverse transcription-polymerase chain reaction (RT­PCR) was used to amplify the N- and C-terminal fragments (ZHX1­N and ZHX1­C, respectively) of the hZHX1 gene. The two PCR fragments were cloned into the pEASY-T1 vector and subcloned into the pcDNA3 plasmid to generate a recombinant pcDNA3­ZHX1 plasmid. Following identification by enzyme digestion and DNA sequencing, the recombinant pcDNA3­ZHX1 plasmid was transfected into SMMC-7721 cells. The level of ZHX1 expression was detected by RT-PCR and western blot analysis. Cell growth curve assays were used to evaluate the effect of ZHX1 on cell proliferation. Moreover, the differential expression of ZHX1 between cancer and adjacent cirrhotic liver tissue was investigated by quantitative PCR (qPCR). Enzyme digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid, pcDNA3­ZHX1. qPCR and western blot analysis demonstrated that ZHX1 was efficiently expressed in SMMC-7721 cells and overexpression of ZHX1 may inhibit the proliferation of SMMC-7721 cells. In addition, reduced ZHX1 expression is widespread among cancer tissues from HCC patients. In conclusion, a recombinant eukaryotic expression plasmid, pcDNA3­ZHX1, was successfully constructed. In addition, the current results indicate that a low expression of ZHX1 may be responsible for hepatocarcinogenesis.

Protein phosphatase magnesium-dependent 1δ (PPM1D) mRNA expression is a prognosis marker for hepatocellular carcinoma.

BACKGROUND: Protein phosphatase magnesium-dependent 1δ (PPM1D) is an oncogene, overexpressed in many solid tumors, including ovarian cancer and breast cancer. The current study examined the expression and the prognostic value of PPM1D mRNA in human hepatocellular carcinoma (HCC). METHODS: Total RNA was extracted from 86 HCC and paired non-cancerous liver tissues. PPM1D mRNA expression was determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR). Immunohistochemistry assay was used to verify the expression of ppm1d protein in the HCC and non-cancerous liver tissues. HCC patients were grouped according to PPM1D mRNA expression with the average PPM1D mRNA level in non-cancerous liver tissue samples as the cut-off. Correlations between clinicopathologic variables, overall survival and PPM1D mRNA expression were analyzed. FINDINGS: PPM1D mRNA was significantly higher in HCC than in the paired non-cancerous tissue (p<0.01). This was confirmed by ppm1d staining. 56 patients were classified as high expression group and the other 30 patients were categorized as low expression group. There were significant differences between the two groups in term of alpha-fetoprotein (α-FP) level (p<0.01), tumor size (p<0.01), TNM stage (p<0.01), recurrence incidence (p<0.01) and family history of liver cancer (p<0.01). The current study failed to find significant differences between the two groups in the following clinical characteristics: age, gender, portal vein invasion, lymphnode metastasis, hepatitis B virus (HBV) infection and alcohol intake. Survival time of high expression group was significantly shorter than that of low expression group (median survival, 13 months and 32 months, respectively, p<0.01). CONCLUSION: Up-regulation of PPM1D mRNA was associated with progressive pathological feature and poor prognosis in HCC patients. PPM1D mRNA may serve as a prognostic marker in HCC.

Activation of calpains mediates early lung neutrophilic inflammation in ventilator-induced lung injury.

Lung inflammatory responses in the absence of infection are considered to be one of primary mechanisms of ventilator-induced lung injury. Here, we determined the role of calpain in the pathogenesis of lung inflammation attributable to mechanical ventilation. Male C57BL/6J mice were subjected to high (28 ml/kg) tidal volume ventilation for 2 h in the absence and presence of calpain inhibitor I (10 mg/kg). To address the isoform-specific functions of calpain 1 and calpain 2 during mechanical ventilation, we utilized a liposome-based delivery system to introduce small interfering RNAs targeting each isoform in pulmonary vasculature in vivo. Mechanical ventilation with high tidal volume induced rapid (within minutes) and persistent calpain activation and lung inflammation as evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition significantly attenuated these inflammatory responses caused by lung hyperinflation. Depletion of calpain 1 or calpain 2 had a protective effect against ventilator-induced lung inflammatory responses. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition also reduced endothelial nitric oxide (NO) synthase (NOS-3)-mediated NO production and subsequent ICAM-1 phosphorylation following high tidal volume ventilation. These results suggest that calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1 phosphorylation and neutrophil recruitment. Inhibition of calpain activation may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.

[The structure and viability analysis of corneal epithelial flap in the rabbit cornea after Epi-LASIK].

OBJECTIVE: To evaluate the changes of ultrastructure and viability in the rabbit corneal epithelial flap after Epi-LASIK (epiploic laser in-situ keratomileusis) surgery and its effect on keratocyte apoptosis and proliferation of peripheral corneal epithelium (out of corneal epithelial flap). METHODS: Fifty-eighty eyes of 29 New Zealand rabbits were used, Epi-LASIK was performed in 28 eyes and Photorefractive keratectomy (PRK) was carried out in 24 eyes. treated-eyes were randomly divided into four groups and were sacrificed at 1, 3, 5, 7 days after surgery, six eyes without treatment were served as blank controls. Histological structure from The specimens of Epi-LASIK and controls eyes were assessed by light, transmission electron microscopy; epithelial cells viability were assessed by enzyme-histochemistry and Immunohistochemistry staining (proliferating cell nuclear antigen, PCNA) were performed to detect proliferation of peripheral corneal epithelial cells. Apoptotic cells were detected by TUNEL assay (TdT-mediated dUTP nick-end labeling) from the specimens of Epi-LASIK and PRK. RESULTS: The study from Transmission electron microscopy demonstrated that epithelial flap separated by KM5000D type epikeratome retained its typical stratification and integrity and the basement membrane including lamina densa and lamina lucida were compaginated with stroma. The expression of ATP enzyme, G-6-P enzyme from epithelial flap to peripheral epithelium of Epi-LASIK-treated eyes were (79%, 58%, 69%, 86%), (79%, 63%, 77%, 97%) at 1, 3, 5, 7 days after surgery respectively. There was no statistically significant difference in the cell Viability and the number of PCNA in peripheral epithelial cells among four Epi-LASIK groups and control group. At 1 day after surgery, no difference in TUNEL positive cells were seen between specimens of Epi-LASIK (3.429 +/- 1.693) and PRK (3.796 +/- 1.998); At 3, 5, 7 days, there was a significant difference in the number of keratocyte apoptosis in PRK compared to Epi-LASIK specimens. CONCLUSIONS: Epithelial flap separated by KM5000D type epikeratome retained its typical stratification and integrity. The flap keeps high viability and no peripheral epithelial cell proliferation and therefore it may play a role in the inhibition of keratocyte apoptosis.

[Advances on surgeries to correct presbyopia].

Surgeries to correct presbyopia are in its initial stage, Conductive Keratoplasty and LASIK multifocal ablation patterns are newly emerging methods. Multifocal IOL or Accommodative IOL provides new method to compensate accommodation of presbyopes, studies on scleral surgeries to restore accommodation are also in progress.