Royal jelly attenuates nonalcoholic fatty liver disease by inhibiting oxidative stress and regulating the expression of circadian genes in ovariectomized rats.

Nonalcoholic fatty liver disease (NAFLD) has a high incidence in postmenopausal women and is accompanied by insulin resistance, obesity, and dyslipidemia. Royal jelly (RJ), a natural substance derived from hive, possesses numerous health-beneficial properties. Here, we evaluated the effects of RJ (150, 300, and 450 mg kg-1  day-1 , 8 weeks) on NAFLD in ovariectomized (OVX) rats. Based on the results, RJ ameliorated the degree of anxiety, improved serum lipid profile, and attenuated the hepatic steatosis and liver injury in OVX rats. Furthermore, the protective effects of RJ could be attributed to its antioxidant properties, which enhance the levels of hepatic antioxidant enzymes. The qRT-PCR results also suggest that RJ improves the disturbances of circadian genes by downregulating their expression, including that of Per1 and Per 2, in the liver of OVX rats. Altogether, our findings suggest that RJ may be a promising agent for the treatment of NAFLD. PRACTICAL APPLICATIONS: Postmenopausal women are at an increased risk of NAFLD. Currently, there are no licensed therapies for NAFLD. Although hormone replacement therapy (HRT) is reported to inhibit the development of NAFLD, it causes unexpected adverse effects. As HRT is controversial, the use of natural supplements to counteract the detrimental effects of menopause has recently attracted more attention. RJ is a natural product secreted from the hypopharyngeal and mandibular glands of worker bees. The present study illustrates the protective effect of the natural product, RJ, and its underlying mechanisms on NAFLD. This is the first study to assess the effect of RJ on NAFLD under estrogen deficiency. Such findings contribute to the further utilization of RJ, which might serve as a promising therapeutic option and natural food for the treatment of NAFLD.

Ethanol Extract of Chinese Propolis Attenuates Early Diabetic Retinopathy by Protecting the Blood-Retinal Barrier in Streptozotocin-Induced Diabetic Rats.

Propolis has been shown to reduce the level of blood glucose and suppress the histopathological changes in diabetics. However, it still remains unknown if propolis has a similar effect on diabetic retinopathy (DR). Our study aimed to evaluate the effect of the ethanol extract of Chinese propolis (EECP) on early DR in streptozotocin (STZ)-induced diabetic rats. EECP was given to diabetic rats by oral intubation for 12 weeks. The concentrations of fasting blood glucose (FBG), glycated hemoglobin (HbA1c), malondialdehyde (MDA), reactive oxygen species (ROS), and reactive nitrogen species (RNS) were measured. Pathological examinations, including hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and immunofluorescence, were also conducted to provide further evidence of EECP's effect on early DR. EECP was able to attenuate diabetes via directly decreasing the levels of FBG and HbA1c, which also resulted in the reduction of MDA, ROS, and RNS. Furthermore, EECP could protect against the damages of photoreceptor cells, as well as retinal thickening. And the inhibition of blood-retinal barrier (BRB) leakage was also observed in EECP-treated diabetic rats, along with the inhibition the loss of tight junction proteins (occludin, ZO-1). These results suggest that EECP has an ameliorating effect on early DR by inhibition of blood-retinal barrier breakdown. PRACTICAL APPLICATION: This study sheds light on the protective effect of the ethanol extract of Chinese propolis on early diabetic retinopathy and the molecular actions underlying the inhibition of blood-retinal barrier breakdown. Our study suggests that ethanol extract of Chinese propolis can be considered as a potential therapeutic agent in the treatment of early diabetic retinopathy.

Biodistribution and preparation of technetium-99m-labeled D-D3 monoclonal antibody against pro-gastrin-releasing peptide (31₋98) in mice.

BACKGROUND: We previously reported that iodine-131((131)I)-labeled anti-pro-gastrin-releasing peptide (ProGRP(31-98)) monoclonal antibody D-D3 could selectively accumulate in the tumor sites of nude mice bearing small cell lung cancer (SCLC) xenografts. However, (131)I-D-D3 was cleared slowly from the body, and the best radioimmunoimaging time for SCLC was 72 - 96 hours after injection. The aims of this study were to radiolabel anti-ProGRP(31-98) D-D3 monoclonal antibody with technetium-99m ((99m)Tc) and to investigate the biodistribution of this antibody in healthy ICR mice. METHODS: D-D3 was labeled with (99m)Tc via the 2-mercaptoethanol reduction method. (99m)Tc-D-D3 was purified by the gel column separation method. The labeling efficiency and radiochemical purity were measured by thin-layer chromatography. The immunological activity of (99m)Tc-D-D3 was determined with cell conjugation assays. (99m)Tc-D-D3 was injected into healthy ICR mice via a tail vein, and all the healthy ICR mice were sacrificed by cervical dislocation at a designated time. Then, the blood and major organs were removed and weighed, and counted in a gamma scintillation counter to determine the percentage of the injected dose per gram (%ID/g). RESULTS: The labeling rate and the radiochemical purity of (99m)Tc-D-D3 were (73.87 ± 2.89)% and (94.13 ± 4.49)%, respectively. The immunobinding rates of (99m)Tc-D-D3 to the human small cell lung cancer NCI-H446 cell line and lung adenocarcinoma A549 cell line were (81.2 ± 2.37)% and (24.3 ± 1.46)%, respectively. The distribution data of normal ICR mice demonstrated that (99m)Tc-D-D3 was mainly distributed in the liver, kidney and lung, and less in the brain tissue and muscle. CONCLUSIONS: (99m)Tc-D-D3 antibody not only had high radiochemical purity, but also had good stability both in vitro and in vivo, and maintained good immunological activity. (99m)Tc-D-D3 was metabolized mainly in the kidney and liver, and the blood radioactivity decreased rapidly. Thus, (99m)Tc-D-D3 is conducive to the radioimmunoimaging of SCLC.

Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro.

BACKGROUND: The sodium-iodide symporter (NIS) protein can mediate the active radioiodine uptake. The human telomerase reverse transcriptase (hTERT) promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting. We constructed a recombinant adenovirus containing the human sodium iodide symporter (hNIS) gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of (131)I in a lung cancer cell line in vitro. METHODS: The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL*-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack. The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system. A positive control adenovirus Ad-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly. A549 cells were transduced with recombinant adenoviruses. (125)I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function. Toxic effects of (131)I on tumor cells were studied by in vitro clonogenic assay. RESULTS: We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter. When infected with recombinant adenovirus constructs expressing hNIS directed by hTERT- and CMV-promoters (Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively), the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30-fold compared to the control parental cells, respectively. The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate (NaClO4). The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% (Ad-CMV-hNIS) and 30% (Ad-hTERT-hNIS) of the control group after receiving radioiodide for 7 hours (P < 0.001). CONCLUSION: Our preliminary study indicates that an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter has the potential to become an effective wide-spectrum yet highly specific anti-cancer strategy.

[Combinational effects of bcl-2 antisense oligodeoxynucleotide and Rituximab on proliferation and apoptosis of B-lymphoma Raji cells].

AIM: To study the combinational effects of bcl-2 antisense oligodeoxynucleotide (bcl-2 ASODN) and Rituximab (anti-CD20 monoclonal antibody) on proliferation and apoptosis of B-lymphoma Raji cells in vitro and in vivo, and to explore the possible mechanisms. METHODS: After bcl-2 ASODN and Rituximab treating on Raji cells respectively or cooperatively, the proliferation of Raji cells, the expression of bcl-2 protein, the apoptosis and the expression of bcl-2 mRNA were detected by MTT assay, flow cytometry (FCM) and RT-PCR, respectively. The combinational antitumor activity of bcl-2 ASODN and Rituximab was evaluated in BALB/c nude mice bearing B-lymphoma inoculated with Raji cells. RESULTS: 5-30 micromol/L bcl-2 ASODN and 1-16 mg/L Rituximab could inhibit Raji cells' growth, induce apoptosis and decrease the expression of bcl-2 protein and bcl-2 mRNA alone. But there was much more effective inhibition of growth and induction of apoptosis by their combination than alone (P<0.01). In tumor bearing mice, combination of bcl-2 ASODN and Rituximab showed a significant inhibitory effect on the growth of transplanted B-lymphoma, resulting in a statistically significant difference compared with alone (P<0.01). CONCLUSION: It has been found that bcl-2 ASODN combined with Rituximab could significantly inhibit the growth of Raji cells and induce their apoptosis via reinforcing specific down regulation of bcl-2 protein and bcl-2 mRNA expression.

[Construction and screening of the specific Fab phage antibody library against Raji cell strain].

AIM: To construct and screen the specific Fab phage antibody library against human Raji cell strain in B-lymphoma. METHODS: BALB/c mice were immunized with Raji cells, and the antibody light chain kappa genes and heavy chain genes Fd from the spleen cells were amplified by RT-PCR. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the light chain kappa genes and heavy chain genes Fd were inserted into the phagemid vector pComb3H-SS successively and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Raji cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. The specific antibodies against Raji cells were obtained after selected with Raji cells. The binding activity with antigens was identified by ELISA and the positive clones were sequenced. RESULTS: The Fab phage antibody library with 2.18 x 10(7) volume was constructed and eight positive clones which specifically recognized Raji cell strain were isolated. Sequence analysis of the two positive clones showed that the variable heavy domains (VH) and variable light domains (VL) were highly homologous with the registered murine Ig heavy chain V region sequences and kappa light chain sequences, respectively. CONCLUSION: Fab phage antibody library was successfully constructed and specific antibodies against membrane antigens in Raji cells were obtained, which will provide an experimental foundation for the further investigation of B-lymphoma immunotherapy.