Poloxamer 407/188 Binary Thermosensitive Gel as a Moxidectin Delivery System: In Vitro Release and In Vivo Evaluation.

Moxidectin (MXD) is an antiparasitic drug used extensively in veterinary clinics. In this study, to develop a new formulation of MXD, a thermosensitive gel of MXD (MXD-TG) was prepared based on poloxamer 407/188. Furthermore, the gelation temperature, the stability, in vitro release kinetics and in vivo pharmacokinetics of MXD-TG were evaluated. The results showed that the gelation temperature was approximately 27 °C. MXD-TG was physically stable and can be released continuously for more than 96 h in vitro. The Korsmeyer−Peppas model provided the best fit to the release kinetics, and the release mechanism followed a diffusive erosion style. MXD-TG was released persistently for over 70 days in sheep. Part of pharmacokinetic parameters had a difference in female and male sheep (p < 0.05). It was concluded that MXD-TG had a good stability, and its release followed the characteristics of a diffusive erosion style in vitro and a sustained release pattern in vivo.

Crocetin protects against fulminant hepatic failure induced by lipopolysaccharide/D-galactosamine by decreasing apoptosis, inflammation and oxidative stress in a rat model.

Fulminant hepatic failure (FHF) is a clinical syndrome characterized by sudden and severe liver dysfunction. Apoptosis and inflammation are essential for the pathogenesis of FHF. Crocetin, the major component present in saffron, has been reported to possess anti-inflammatory and antioxidant functions; however, its role in FHF is poorly understood. The aim of this study was to explore the protective effects of crocetin against lipopolysac§§charide (LPS)/D-galactosamine (D-GalN)-induced FHF and the underlying mechanisms in a rat model. For the in vivo study, rats were assigned to the LPS/D-GalN group or to the crocetin pre-treatment+LPS/D- GalN group. Each group was then further divided according to the different LPS/D-GalN treatment times of 0, 6, 12 or 48 h. The results demonstrated that crocetin pre-treatment efficiently protected against LPS/D-GalN-induced FHF by improving liver tissue morphology, reducing total bilirubin generation and decreasing the activities of alanine transaminase and aspartate aminotransferase. Moreover, crocetin pre-treatment significantly decreased hepatocyte apoptosis, p53 mRNA expression and the expression of proteins in the caspase family and the Bcl-2 pro-apoptotic family following LPS/D-GalN treatment. Furthermore, crocetin also decreased the secretion of pro-inflammatory cytokines in the serum and in the liver via suppression of NF-κB activation, and also suppressed hepatic oxidative stress. In conclusion, crocetin protected against LPS/D-GalN-induced FHF and inhibited apoptosis, inflammation and oxidative stress. The underlying mechanisms may be related to the regulation of apoptotic proteins in the caspase family and the Bcl-2 family, as well as the modulation of NF-κB expression. Therefore, crocetin may be used as a novel therapy for preventing FHF.

[Crocetin promotes autophagy in injured rat hepatocytes induced by lipopolysaccharide and D-galactosamine in vitro].

OBJECTIVE: To observe the effect of crocetin on autophagy in rat hepatocytes exposed to lipopolysaccharide (LPS) and D-galactosamine (D-gal) and explore the mechanism. METHODS: Cultured rat hepatocytes were exposed to LPS (1 mg/L) and Dgal (60 mg/L) to induce cell injury and treated with crocetin, 3MA, or crocetin+3MA. Twelve hours after the treatments, the cells were examined for levels of ALT, AST and LDH in the supernatant using ELISA. LC3 fluorescence in the cells following immunofluorescence staining was observed using fluorescence microscopy. Autophagosomes in the cells were observed by transmission electron microscopy, and the cellular expressions of LC3, p62 and SIRT1 were detected using Western blotting. RESULTS: The levels of ALT, AST and LDH in the hepatocytes were elevated after LPS- and D-gal-induced injury, reached the highest levels after 3MA treatment, but were decreased significantly by crocetin treatment. LC3 fluorescence increased obviously in the injured hepatoctyes, and the increment was the most obvious in crocetin-treated cells; LC3 fluorescence was decreased significantly after 3MA treatment. Cell injury induced obvious increase in autophagy in the hepatocytes, and the number of autophagosomes increased significantly after crocetin treatment but was reduced significantly after 3MA treatment. The cell injury caused an obvious up-regulation of LC3 and SIRT1 expression and down-regulated p62 expression. LC3 and SIRT1 expression levels were the highest and the expression of p62 was the lowest in cells with crocetin treatment. 3MA treatment significantly reduced the expression of LC3 and SIRT1 and increased the expression of p62 in the injured cells. CONCLUSIONS: Autophagy is increased in injured rat hepatocytes, and crocetin can promote autophagy in the injured cells to reduce further cell injury.

NADH protect against radiation enteritis by enhancing autophagy and inhibiting inflammation through PI3K/AKT pathway.

Radiotherapy is an important method for cancer treatment but it has serious side-effects at high doses. One of the greatest challenges in radiotherapy is that radiation affects both healthy tissue and cancer tissues. For abdominal or pelvic lesions, the bowel is the most easily injured by irradiation. Radiation may cause radiation enteritis, intestinal inflammatory infiltration, or intestinal perforation. Coenzyme NADH involves in energy metabolism and transportation of nucleic acid, proteins and carbohydrates. In our study, NADH was used to protect the intestinal wall from irradiation injury in IEC-6 normal intestinal epithelial cells. By flow cytometry, we found that NADH can inhibit the cell death and the producing of reactive oxygen species (ROS). The immunofluorescence assay showed that cell autophagy was increased in the NADH group. Western blot data indicated that NADH promoted the microtubule associated protein 1A/1B-light chain 3(LC3)-I to LC3II and the expression of IL-1ß and TNFα decreased in a dose dependent manner. Interestingly, a specific PI3K/AKT inhibitor (3MA) decreased the expression of inflammatory factors. In the animal experiment, after 12 Gy radiation, there were less TNFα and more LC3II in the RT+NADH group than that of RT group. Compared with the mock, there was no significant damage in the NADH group. Thus, our study provides the evidence that NADH may protect against radiation enteritis by suppressing inflammation and enhancing autophagy through PI3K/AKT pathway in normal intestinal cells.

miR-224 suppresses HBV replication posttranscriptionally through inhibiting SIRT1-mediated autophagy.

Hepatitis B virus (HBV) enters the host and successfully completes replication by using several mechanisms, including autophagy. However, previous studies revealed that microRNAs (miRNAs) widely participate in regulation of various cellular processes, such as autophagy and viral replication. Hence, the purpose of this study was to investigate the role of miR-224 in HBV infection and to determine whether its role depended on the miR-224/SIRT1/autophagy axis. Our results show that secretions of HBeAg and HBsAg, and HBV replication significantly declined in Huh7-1.3 cells, established by transfecting recombinant pcDNA 3.0-1.3 mer containing the 1.3 mer fragment of HBV genomic DNA,with miR-224 mimic transfection as compared to the Huh7-1.3 group. Moreover, it was discovered that HBV could induce autophagy, while miR-224 inhibited autophagy caused by HBV. Additionally, miR-224 could suppress SIRT1, LC3 expression, and facilitate p62 expression. SIRT1 was identified as the target gene of miR-224 and down-regulation of SIRT1 via miR-224 or si-SIRT1 transfected treatment in Huh7-1.3 cells repressed LC3 expression and enhanced p62 expression. In conclusion, these results suggest that miR-224 might hinder HBV replication through attenuating SIRT1-mediated autophagy, thereby these findings open a new avenue for the treatment of HBV infection.

High expression of Fibronectin 1 suppresses apoptosis through the NF-κB pathway and is associated with migration in nasopharyngeal carcinoma.

Fibronectin 1 (FN1) is a member of the glycoprotein family located on chromosome 2q35. It has been reported that FN1 is upregulated in many tumors, and its expression is negatively related to the prognosis and survival of cancer patients. Through data analysis, we found that FN1 is upregulated in nasopharyngeal carcinoma (NPC). This study aimed to investigate how FN1 expression affects NPC cell behavior. In this study, we downregulated FN1 in two NPC cell lines, 5-8F (EBV-) and C666-1 (EBV+), and evaluated invasion, migration and apoptosis. FN1 promoted migration and invasion by upregulating MMP9 and MMP2 expression; the NF-κB/P65 signaling pathway was also affected by FN1. FN1 suppressed apoptosis in NPC cells by upregulating BCL2 and increasing the nuclear localization of P65, both by inducing cytosolic accumulation and nuclear translocation, but FN1 expression was not reduced when the NF-κB/P65 pathway was inhibited in the negative control (NC) group. Compared with NC cells, shFN1 cells showed little change in apoptosis when the NF-κB/P65 pathway was activated by LPS. These results suggest that FN1 regulates apoptosis though P65 in the NF-κB pathway. Our results show that FN1 plays an important role in NPC cells and is a potential target for NPC treatment.

Complications of chemoembolization for hepatic neoplasms.

OBJECTIVE: To present a safe and effective approach to chemoembolization for hepatic neoplasms, and to discuss the complications of chemoembolization and ways of avoiding them. METHODS: The techniques and experience described herein are based on clinical practice at Yichang Central People's Hospital, Yichang, Hubei, China, where over 200 chemoembolization procedures are performed yearly, and on the results of an intensive review of 1054 chemoembolization procedures performed between July 1997 and December 2005. RESULTS: There were complications as follow: 5 cases with celiac artery branch embolization, gastric uptake in 4, 6 with gallbladder uptake and infarction, splenic uptake and infarction in 8, liver infarction and abscess formation in 3, and hepatorenal syndrome in 4, liver rupture in 2, lung uptake in 6, and spinal cord injury in 2 cases. CONCLUSION: There are numerous potential errors and complications associated with chemoembolization for unresectable liver tumors. A good understanding of the congenital and acquired variations of arterial anatomy that may be seen supplying the liver is required.

X-ray induced L02 cells damage rescued by new anti-oxidant NADH.

AIM: To explore molecular mechanism of nicotinamide adenine dinucleotide (NADH) antagonization against X-ray induced L02 cells damage. METHODS: L02 liver cells were cultured in RPMI 1640, exposed to X-ray irradiation and continued to culture in the presence or absence of NADH. Cellular viability was analyzed by routine MTT methods. The percent age of apoptotic cells and positive expressions of p53, bax and bcl-2, fas, fasL proteins were determined by FCM. Level of intracellular ROS was determined by confocal microscope scanning. Morphological change was detected by scanning electron micrograph. RESULTS: The viability of L02 cells was decreased with increasing dose of X-ray irradiation. NADH could not only eliminate the apoptosis induced by X-ray irradiation, but also up-regulate expression of bcl-2 protein and down-regulate expression of p53, bax, fas and fasL proteins (P<0.05). At the same time, NADH could reduce level of intracellular ROS in radiated L02 cells. CONCLUSION: NADH has marked anti-radiation effect, its mechanism may be associated with up-regulation of bcl-2 expression and down-regulation of p53, bax fas and fasL expression, as well as decline of intracellular ROS. However, further investigation of its mechanism is worthwhile.

Protective effect of coenzyme I on mouse hematopoietic system against radiation.

OBJECTIVE: To study the protective effect of NADH on the hematopoietic system of mice against radiation. METHODS: Sixty mice were randomized into 3 groups (20/group), Group I serving as normal control group. Three days before exposure to single-dose (6.0 Gy) whole body 60Co gamma-ray irradiation in the other 2 groups, the mice in Group II received injections with 0.9% NaCl and those in Group III with intraperitoneal NADH (10 mg/kg, twice daily). The peripheral white blood cells count was carried out in the blood samples from the mouse tail vein at different time after the exposure, and the bone marrow smears were prepared with routine staining to examine the number of the granulocytes and determine their mitotic index 24 h after radiation. DNA fragmentation in mouse bone marrow cells was detected by DNA electrophoresis 24 and 48 h after irradiation. RESULTS: The white blood cell count was significantly higher in mice with coenzyme I treatment before exposure to the irradiation. NADH also increased the number of the granulocytes and their mitotic index without inhibiting DNA fragmentation in the bone marrow cells as observed 24 and 48 h after the irradiation. CONCLUSION: NADH can markedly prevent the damages to the hematopoietic system in mice exposed to irradiation, but the mechanism does not involve the inhibition of bone marrow cell apoptosis.

[Effects of lipofectin-mediated alpha1,4Gal T antisense oligonucleotide on human glioma cell line SWO-38].

OBJECTIVE: To study the effects of alpha1,4Gal T antisense oligonucleotide mediated by lipofectin on human glioma cell line SWO-38. METHODS: SWO38 glioma cells were exposed to 10 micromol/L alpha1,4Gal T antisense oligonucleotide for 72 h by means of lipofectin transfection, and the growth inhibition of the cells was detected by colony-forming unit assay. Analysis of DNA fragmentation was performed with flow cytometry (FCM) and agarose gel eletrophoresis with Fas protein expression determined by FCM. RESULTS: alpha1,4Gal T antisense oligonucleotide significantly inhibited the growth of glioma cells (P<0.01) and induced apoptosis in SWO-38 cells (P<0.05). Marked up-regulation of Fas protein expression was observed in response to the treatment (P<0.01). CONCLUSION: alpha1,4Gal T antisense oligonucleotide can significantly inhibit SWO-38 cell proliferation and induce cellular apoptosis, the mechanism of which may involve up-regulated Fas expression.

[Effect of NADH against liver cell line L02 apoptosis induced by UVB irradiation].

OBJECTIVE: To study the molecular mechanism behind the effect of reduced nicotinamide adenine dinucleotide (NADH) against apoptosis of liver cell line L02 induced by ultraviolet B (UVB) exposure. METHODS: L02 liver cells were exposed to UVB irradiation (200 J/m(2)) followed by a 24-hour culture in the presence or absence of NADH (400 microgram/ml). The degraded fragments of DNA in the cells were observed by agarose electrophoresis, the cell apoptosis rate determined by Annexin/V PI staining and p53, Bax and Bcl-2 proteins expression detected by flow cytometry. RESULT: NADH not only inhibited the apoptosis induced by UVB irradiation, but also up-regulated the expression of Bcl-2 protein and down-regulated expressions of p53 and Bax proteins (P<0.01). Repair of DNA strand damage was observed in L02 cells during the incubation time after irradiation. CONCLUSION: NADH significantly inhibits apoptosis induced by UVB irradiation possibly by the mechanism of up-regulating Bcl-2 expression and down-regulating p53 and Bax expressions.

Effect of coenzyme I on apoptosis of liver cell line L02 induced by ischemia-reperfusion injury.

OBJECTIVE: To study the protective effect of coenzyme I (NADH) on normal liver cell line L02 against ischemia-reperfusion injury. METHODS: Cultured L02 cells was divided into 3 groups, namely ischemia-reperfusion group (I/R), ischemia-reperfusion group with NADH pretreatment (NADH+I/R) and control group. Assisted by flow cytometry (FCM), the apoptosis rate and the expression of apoptosis-related proteins (Bcl-2, p16, p21, p53) were detected in L02 cells in the 3 groups. Apoptotic L02 cells were also observed under transmission electron microscope. RESULTS: I/R significantly inhibited L02 cell proliferation and increased their apoptosis rate up to (31.53+/-8.27)% 12 h after I/R. In the presence of NADH, however, the apoptotic rate of the cells was significantly decreased to (12.61+/-2.34)% (P<0.05). FCM indicated that I/R down-regulated the expression of Bcl-2 and up-regulated the expression of p16, p21 and p53 proteins as compared with control group, but NADH acted to the reverse effect. Typical apoptotic features were observed by transmission electron microscope in the cells 12 h after I/R. CONCLUSION: I/R might induce apoptosis in L02 cells by up-regulating the expression of p16, p21, p53 and down-regulating the expression of Bcl-2, and the protective effect of NADH against I/R-induced apoptosis may lie in the expression regulation of the proteins concerned.