We analyzed the anticancer effect and mechanism of the novel indoleamine 2,‍3-dioxygenase 1 (IDO1) inhibitor NLG-919 combined with temozolomide (TMZ) on human glioma cell lines. The anti-tumor activity of NLG-919 and temozolomide after single and combined treatments was detected by MTT assay. Colony formation assay, invasion assay and migration assays were used to detect the effects of NLG-919 and temozolomide alone or in combination on proliferation, invasion and migration of human glioma cells. A flow cytometry assay was used to detect cell apoptosis, cell cycle arrest, reactive oxygen species (ROS) production and mitochondrial membrane potential damage (JC-1). An immunofluorescence assay was used to detect the expression level of IDO1 and HPLC was used to detect the expression level of L-kynurenine (Kyn) to explore the anti-tumor mechanism of NLG-919 and temozolomide. The results show that NLG-919 had a weak in vitro inhibitory effect compared to that of temozolomide. The IC₅₀ of NLG-919 on U251 cells and U87 after 72 h was 26.9 and 30.7 μmol·L^-1, respectively. However, when NLG-919 was used in combination with temozolomide, its anti-glioma activity was significantly increased. Compared with the single treatment, the combination treatment had a potent ability to inhibit proliferation, invasion and migration of glioma cells. Combination treatment improved the capacity of temozolomide to induce cell cycle arrest and inhibit the growth of glioma cells. NLG-919 significantly down-regulated the expression and activity of IDO1 in glioma cells, and the inhibitory effect was improved after combination with temozolomide, and effectively blocked the production of Kyn through the metabolism of L-tryptophan (Trp). In conclusion, the IDO1 inhibitor NLG-919 and temozolomide showed synergistic effects in the anticancer therapy of human glioma cell lines.

AIM:To construct the recombinant eukaryotic expression plasmid pEGFP-C1-SR-A I for the high expression in 293T cells in order to identify functions of savenger receptor-A I(SR-A).METHODS:The primer was designed according to MSR1 cDNA and pEGFP-C1-SR-A I was constructed by standard molecular cloning technique and enzyme digestion.After sequencing,the plasmid was transfected into 293T cells by lipidosome method.The expression of scavenger receptor-A I was identified by RT-PCR and Western blotting.The foam cells were evaluated by the formation of lipid granules in the cells with oil red staining.Cell adhesion was analyzed by cell adhesion assay.RESULTS:24 h after transfection,SR-A I mRNA was highly expressed and the high level of the protein was detected.The ratio of foam cell formation was doubled,the efficacy of cell adhesion was enhanced two times compared to the control group and the empty vector group.CONCLUSION:The recombinant eukaryotic expression plasmid has been constructed successfully with enhancing the function of uptake ox-LDL and adhesion in 293T cells by overexpression of SR-A I.

AIM: To assess the differentiation of rat mesenchymal stem cell (MSC) in the microenvironment of retinitis pigmentosa(RP) induced by the administration of sodium iodate. METHODS: In vitro cultured Lewis rat MSC were injected into the subretinal space of NaIO3 induced RP rat eyes (30g/L NaIO3 100mg/kg). To observe the trace and differentiation of MSC by immuno-fluorescent method successively in 5 weeks after the surgery.RESULTS: The majority of the transplanted cells stay in retinal pigment epithelium(RPE) layer and cones and rods layer. From the 2nd week after transplantation, the engrafted MSC expressed PCK and rhodopsin under fluorescent microscope.CONCLUSION: MSC can survive mainly in the outer layer of retina in the microenvironment of RP and differentiate forward the RPE cell and photoreceptor.

AIM: To discuss the clinical applications of methods to localize nasal cut ends and the effects of Z-plasty in the surgeries for lower eyelid longitudinal laceration combined with lower lacrimal canaliculi disruption.
METHODS: From September, 2010 to October, 2013, a total of 37 patients ( 37 eyes ) with lower eyelid longitudinal laceration combined with lower lacrimal canaliculi disruption were operated for anastomosis of lacrimal canaliculi disruption and suture of lower eyelid longitudinal. Different methods to search for the nasal cut ends of lacerated lacrimal canaliculi, such as “under a microscope directly”, “guided by probing needle” and“pigtail curved probe”. Then, to repair lower eyelid longitudinal laceration with Z-plasty transposition flaps. Follow up was 3mo~2a after operation.
RESULTS: All nasal cut ends could be found successfully on 37 patients;Lacrimal duct unobstructed in 31 patients (83. 8%), improved in 5 patients (13. 5%), invalid in 1 patient (2. 7%),the overall successful rate was 97. 3%; the eyelids repair was satisfactory, small scars, the appearance and function was normal.
CONCLUSION: The nasal cut ends can be found successfully by “directly under a microscope”, “guided by probing needle” and“pigtail curved probe”;the effect of silicone drainage tube used as lacrimal canaliculi bracket is satisfactory; most patients gained excellent recovery for both appearance and function after Z-plasty.

Background Congenital fibrosis of extraocular muscles (CFEOM) affects patient' s appearance and quality of life,and no effective treatment for this disease is available.Imaging study is helpful for exploring the pathogenesis of CFEOM.Objective This study was to describe the characteristics of CFEOM associated with limb movement disorder using magnetic resonance imaging (MRI).Methods A family with CFEOM associated with limb movement disorder was investigated in Renmin Hospital of Wuhan University.Disease history was collected and the pedigree was investigated.Ophthalmologic examinations,including corrected visual acuity,refractive error,slitlamp examination,ophthalmoscopic examination,force of levator palpebrae superioris,ocular movement,eye position,forced duction test,and bell phenomenon examination,were performed.Ocular orbital and cranial MRI was performed in 4 CFEOM patients and 10 normal subjects to compare the structures of the extraocular muscles,motor nerve and cranium.Oral informed consent was obtained from each patient prior to any medical examination.Results A total of 1 1 members from 3 generations were investigated in this study,presenting with 4 cases of disease.The mode of inheritance of this family complied with the Mendelian autosomal dominant inheritance law.Clinical signs included disturbance of eye movement,deviation of eye position,ptosis,lack of Bell sign and positive reaction of passive pull test.In addition,unstable gait,improper body limb alignment,dysphasia and mental retardation were ohserved in 1 patient,which coincided with the diagnostic criteria of type 3 CFEOM.MRI results demonstrated that the levator palpebrae superioris,superior rectus and superior oblique muscle were clearly thinner,and the medial rectus,lateral rectus,inferior rectus muscle were thinning in the patients,showing significant differences in comparison with the normal controls(P＜O.05).The oculomotor and abducens nerves became thinner and even absent in the patients.Cranial MRI showed that Ⅲ-3 in the pedigree with callosum was shorter than that of the normal controls,suggesting that patient suffered from corpus callosum hypoplasia.Meanwhile,cranial MRI indicated the presence of cerebellar hypoplasia and the expansion of the fourth ventricle.Conclusions MRI demonstrates consistent abnormalities of the oculomotor nerves and abducens nerves in the affected individuals in this CFEOM 3 family,and some affected members exhibit two types of central nervous system abnormalities-corpus callosum and cerebellar hypoplasia.These findings suggest that CFEOM 3 is primarily a neuronal disease.

OBJECTIVETo establish an efficient and stable method for protein extraction of Streptococcus mutans.

METHODSThe collected bacteria were treated by freeze-thaw and ultrasonic (method 1), ultrasonic (method 2), boiling (method 3), boiling and ultrasonic (method 4), respectively. The index such as state of bacteria broken, concentration of extracted protein and SDS-PAGE of protein were employed to evaluate the effects of above four methods.

RESULTSBeside the method 3, the other three methods could break the bacteria effectively, of which ultrasonic was the key factor. The pattern of SDS-PAGE which treated by method 1, method 2 and method 4 was almost same, but method 1 resulted in the best abundance. There was significantly difference among the four protein concentration extracted by four methods (P < 0.05). All methods exhibited good stability and reproducibility.

CONCLUSIONMethod of freeze-thaw and ultrasonic resulted in an efficient proteins extraction of Streptococcus mutans which demonstrated good stability and reproducibility and easy to handle.

Bacterial Proteins ; Reproducibility of Results ; Streptococcus mutans

To investigate the effect of losartan on the axis of prolylcarboxypeptidase (PRCP)--kallikrein of the two-kidney, one-clipped (2K1C) hypertensives rats, and explore the novel protection mechanism of losartan on the kidney. Sprague-Dawley (SD) rats were used to develop the 2K1C hypertensive rats. Then, the rats were treated with prazosin (5 mg x kg(-1) x d(-1)) or losartan (5, 15 and 45 mg x kg(-1) x d(-1)) or vehicle, separately. At the same time, the blood pressures were observed. After treated for four weeks, the ratio of right kidney weight and body weight, the change of glomerular morphology, and K+, Na+, creatinine and blood urea nitrogen (BUN) of the serum were used for evaluation of kidney. The expressions of PRCP mRNA in the kidneys were determined by RT-PCR. The protein levels of PRCP, tissue kallikrein, plasma kallikrein, TGF-beta1 in kidney or plasma were measured by Western blotting. Results showed that the changes of body weight and kidney weight ratio, glomerular fibrosis degree and the biochemistrical index of serum induced by hypertension were relieved when the hypertensive rats treated with losartan for four weeks. Meanwhile, treatment of losartan also significantly decreased expression of TGF-beta1 and increased expressions of PRCP, plasma kallikrein and tissue kallikrein. The protective effects of losartan on the kidney of 2K1C hypertensive rats are activation of the axis of PRCP-kallikrein and reducing the expression of TGF-beta1.
Animals ; Antihypertensive Agents ; pharmacology ; Blood Pressure ; drug effects ; Carboxypeptidases ; genetics ; metabolism ; Hypertension, Renovascular ; metabolism ; pathology ; physiopathology ; Kallikreins ; blood ; metabolism ; Kidney ; metabolism ; pathology ; Kidney Glomerulus ; pathology ; Losartan ; pharmacology ; Male ; Organ Size ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; blood ; metabolism

Objective To evaluate the therapeutic effect and security of triple antiplatelet with cilostazol in the elderly after drug-eluting stent implantation and compare it with double antiplatelet treatment. Methods 234 elderly patients with coronary disease were randomly divided into two groups.118 cases in the triple antiplatelet group were treated with clopidogrel (300 or 600 mg/d) and aspirin(100 mg/d) in addition with cilostazol(200mg/d) from pre surgery to 6 month after surgery,then received double antiplatelet treatment.116 cases in the double antiplatelet group were treated with Aspirin(100 mg/d) and clopidogrel(300 or 600 mg/d),then clopidogrel was ceased after 1 year and used only Aspirin. The main parameters during follow up included all-cause death,major adverse cardiovascular events (MACE) and major adverse cardiac and cerebrovascular event (MACCE),the secondary parameters during follow- up were recurrence of angina pectoris,myocardial infarction,revascularization and hemorrhage within 2 years. Results The recurrence of angina pectoris and revascularization were found in 1 case (0.85％) and 1 case(0.85％) respectively in the triple antiplatelet group,while 8 cases(6.90％) and 8 cases (6.90％) in the double antiplatelet group,with significant difference between the two groups(both x2 =4.27,P＜0.05).All cause death,myocardial infarction,cerebral apoplexy and hemorrhage were not found in the triple antiplatelet group,while 1 case of death,1 case with myocardial infarction,1 case with apoplexy and no hemorrhage appeared in the double antiplatelet group,with no significant difference between the two groups(P＞0.05).Conclusions The triple antiplatelet added with cilostazol in the elderly after drug eluting stent implantation may decrease the recurrence of angina pectoris and revascularization with higher security.

Objective To study the clinical and imaging features of patients with bone metastases from breast cancer and identify the factors related to the incidence of bone metastases. Methods Three hundred and thirty-four patients with breast cancer were recruited into this study. Whole-body 99Tcm-methylene disphosphonate (MDP) bone scan, clinical staging, pathological, immunohistochemical and serological test results were analyzed retrospectively. χ2 test was used for statistical analysis. Results The incidence rate of bone metastases for patients with and without lymph node metastases was 71% (152/214) and 22. 5% (27/120), respectively (χ2 =72.80, P =0.000). The incidence rate of bone metastases from infiltrated non-specified and specified breast cancer was 69% (203/294) and 41.7% (5/12), respectively (χ2 =3. 97, P=0.046). Alkaline phosphatase (ALP) was elevated in 28.5% (51/179) and 14.9%(11/74) of patients with and without bone metastases, respectively (χ2 = 5. 25, P = 0.022 ). Carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 15-3, CA125, CA19-9 increased in 68.7% ( 123/179) and 27.0% (20/74) of patients with and without bone metastases, respectively (χ2 = 37. 03, P =0. 000). Conclusions The incidence of bone metastases from breast cancer is correlated to pathological types of primary tumor and lymph node metastases. Bone metastases occurs more frequently in patients with infiltrated, non-specified, primary cancer and with lymph node metastases. Serum ALP, CEA, CA15-3,CA125, CA19-9 might be the tumor makers for early diagnosis of bone metastases from breast cancer.

OBJECTIVETo study the effect of a microRNA-132 antagonist on lithium-pilocarpine-induced status epilepticus (SE) in young Sprague-Dawley (SD) rats.

METHODSForty-five 3-week-old SD rats were randomly and equally divided into epilepticus model group, microRNA-132 antagonist group, and microRNA-132 antagonist negative control group. The young SD rat model of SE was established using lithium-pilocarpine. For the microRNA-132 antagonist group and the negative control group, pretreatment was performed 24 hours before the model establishment. Behavioral observation was performed to assess the latency of SE and success rate of induction of SE. The scale of Lado was used to evaluate the seizure severity. Electroencephalography (EEG) was used to assess the frequency and amplitude of epileptiform discharges. The mortality rate was calculated in each group.

RESULTSThere was no significant difference in the success rate of induction of SE between the three groups (P>0.05). Compared with the microRNA-132 negative control group and the epilepticus model group, the microRNA-132 antagonist group had significantly prolonged SE latency after model establishment (P<0.05), a significantly lower Lado score of seizure (P<0.05), significantly lower frequency and amplitude of epileptiform discharges on EEG (P<0.05), and a slightly reduced mortality rate.

CONCLUSIONSThe treatment with the microRNA-132 antagonist shows an inhibitory effect on the development and progression of lithium-pilocarpine-induced SE in young SD rats. The inhibition of microRNA-132 is likely to be a potential target or direction for drug treatment of SE.

Animals ; Electroencephalography ; Male ; MicroRNAs ; antagonists & inhibitors ; Pilocarpine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Status Epilepticus ; chemically induced ; drug therapy