OBJECTIVE:The preparation of microspheres was optimized by central composite design in order to improve the lung targeting of tetrandrine chitosa microspheres. METHODS:Microspheres were prepared using an emulsion-chemical crosslink technique.Effects of three independent variables i.e. weight percent of tetrandrine and christon, volume percent of water phase and organic phase and christon concentration in aqueous phase were investigated on four response variables.Response variables selected in the research were yield,drug loading, envelop efficiency, mean diameter and span of dispersity.Second-order polynomial and linear equations were fitted to the data,and the resulting equations were used to produce three dimensional response surface graphs,by which optimal experimental conditions were selected. RESULTS:Five response variables were found to be dependent on three independent variables. According to optimal experimental conditions.An optimized formulation contained weight percent of tetrandrine and christon was 61.97%, volume percent of water phase and organic phase was 13.51% and christon concentration in aqueous phase was 2.37%. CONCLUSION:The central composite design can be used to optimazation of preparation formulation,diameter of the microspheres optimized by it can meet the demands of lung-targeting.

OBJECTIVE:To study the lung targeted characteristic of tetrandrine polylactic acid microsphere(TPM).MET_HODS:TPM and tetrandrine injection were injected intravenously into mouse,an RP-HPLC was established to measure the content of TPM in biological samples,and the concentrations of tetrandrine in different tissues of mice were determined and compared.RESULTS:The determinable concentration of tetrandrine in plasma was in the linear range of 0.519～17.000?g/ml(r=0.9 996),the recovery was 97.32%,the mean value of RSD was 4.46%.After the use of TPM,the concentrations of tetrandrine in mouse tissues were significantly higher than tetrandrine injection,and the highest concentration was detected in lungs of mouse.CONCLUSIONS:TPM is distinguishingly lung targeted.

OBJECTIVE:To provide scientific evidence for the microsphere's m etabolism in vivo of lung targeted tetran?drine polylactic acid(TET-PLA)microspheres.METHODS:Blood was sampled from carotid artery after microspheres were injected into rabbit through auricular vein,the concentration of TET in plasma was determined by RP-HPLC method;The pharmacokinetic parameters were obtained by using3p87program.RESULTS:Concentration-time curves of TET micro?spheres were fitted to a2-compartment model with t 1/2? of(286.49?237.55)min and AUC of(810.33?287.49)(?g?min)/ml.CONCLUSION:TET-PLA microspheres show sustained release effects in rabbits.

OBJECTIVE:To establish a method for the rapid contents determination of potassium chloride and calcium chloride in Compound sodium chloride injection. METHODS:After diluted in appropriate way,Compound sodium chloride injection was sampled directly and contents of potassium chloride and calcium chloride were determined simultaneously by ICP-OES. The powder was 1 300 W,plasma gas flow rate was 15 L/min,auxiliary cooling gas flow was 0.2 L/min,atomizer flow rate was 0.8 L/min, the peristaltic pump rate was 0.8 L/min,atomizer pressure was 315 kPa,and the observation was axial observation,analysis spec-tral lines of potassium and calcium were 766.490 nm and 315.887 nm. RESULTS:The linear ranges of potassium and calcium were 1.0-12.0 mg/L (r=0.999 7 and 0.999 9);RSDs of precision and reproducibility tests were lower than 1%;recoveries were 98.5%-100.5%(RSD=0.59%,n=9)and 99.3%-102.3%(RSD=0.98%,n=9). CONCLUSIONS:The method is simple,rapid and simple,and can be used for the quality control of Compound sodium chloride injection.

Objective To explore the effects and mechanisms of urokinase-type plasminogen activator (uPA) on high glucose-induced rat mesangial cells proliferation and phenotype transformation. Methods Rat mesangial cells were cultured and incubated in media containing either 5 mmol/L D-glucose or 30 mmol/L D-glucose with or without addition of wortmannin, or uPA (105 U/L) for different time periods. At the end of the incubation period, mesangial cells proliferation was assessed by MTT assay and flow cytometric analysis. Cyclin-dependent kinase 2 (CDK2) and p27kip1 expression and activation of Akt were evaluated by Western blotting and Akt kinase assay respectively. Furthermore, the expression and distribution of α-SMA were detected with laser confocal microscopy. Results MTT assay and flow cytometric analysis demonstrated that high glucose induced mesangial cells proliferation (P＜0.05) and an incresed proportion of cells in G2/M+S stage after 24 h incubation (P＜0.01), which were attenuated by uPA or wortmannin (P＜0.01). High glucose induced the enhance of Akt activity after 3 h (P＜0.05), and the effect was inhibited by wortmannin or uPA (P＜0.01). High glucose did not alter CDK2 expression (P＞0.05),but significantly inhibited p27kip1 expression (P＜0.05), which was attenuated by wortmannin or uPA (P＜0.01). High glucose induced the up-regulation of α-SMA expression and perinucleus location in mesangial cells after 24 h (P＜0.01), which were alleviated by wortmannin or uPA (P＜0.01). Conclusion uPA up-regulates p27kip1 expression and counteracts high glucose-induced mesangial cells proliferation and phenotype transformation via blocking PI3K-Akt signaling pathway.

Purpose　The aim is to prepare the extract of immunocompetent cell proliferation from human placenta and to try to find out a suitable method of preserving the extract.Methods　The extract was mainly prepared by heat-treating placenta homogenated fluid. Then the activity to stimulate murine splenic lymphocyte proliferation in vitro was done with MTT , exposing　the　extract to　radioisotope 60　Co.Results　The content of protein was 2～3mg per gram placenta measured by Lowry′s Method.The rate to promote cell proliferation was more than 80 percent.The activity lasted a few months after being exposed to radioisotope.Conclusion　The extract prepared by heat-treating not only had high activity, but also had the unique method and good repeatability.This prepared extract as a kind of stable,reliable and remarkably promoting lymphocyte proliferation reaction had the value of development production on broad scale as well as in clinical practice.

ObjectiveTo investigate the effect of Simvastatin on LOX-1 and ROS in NRK52E induced by ox-LDL.MethodsNRK-52E cells were divided into three groups: Control group,ox-LDL group (50 μg/ml ox-LDL) and Simvastatin group (10 μmol/L Simvastatin +50 μg/ml ox-LDL).After incubation for 24 h,the expression of LOX-I was analyzed by Western blotting,and production of reactive oxygen species (ROS) was analyzed with confocal laser scanning microscopy.ResultsNRK-52E expressed LOX-1 at low level,50 μg/ml ox-LDL increased the expression of LOX-1 by 6.80 times.Pre - treatment with Simvastatin decreased LOX-1 expression by 65％.There was little ROS generation in NRK52E cells,50μg/ml ox-LDL promoted the expression of LOX-1 by 4.86.times.Pre - treatment with Simvastatin decreased ROS generation by 60％.ConclusionsSimvastatin upregulate LOX-1 expression and ROS generation induced by Ox-LDL in NRK52E cells.

Objective: To evaluate the role of the MAPK subtypes (p38MAPK, ERK and JNK) in ANG Ⅱ induced apoptosis of cultured human podocytes. Methods: The cultured podocytes were incubated in media containing either vehicle, SB202190(5 ?mol/L, an inhibitor of p38MAPK), PD98059 (1 ?mol/L, an inhibitor of ERK), SP600125 (5 ?mol/L, an inhibitor of JNK), ANG Ⅱ (10 -8 mol/L) with or without SB202190、PD98059 and SP600125 for 18 hours; the cells were assayed for apoptosis by morphologic staining with H 33342 and propidium iodide and DNA fragmentation assays; the cell proteins were probed for phosphorylated MAPKs to determine the activation of specific MAPK subtypes. Results: ANG Ⅱ promoted podocyte apoptosis in a time and dose dependent manner; ANG Ⅱ stimulated p38MAPK, but inhibited JNK; SB202190 inhibited both ANG Ⅱ induced podocyte apoptosis and p38MAPK phosphorylation; Inhibition of ERK by PD98059 had no effect on ANG Ⅱ induced cell apoptosis. Conclusion: ANG Ⅱ induced apoptosis through stimulation of p38MAPK and inhibition of JNK in human podocytes.

Objective To investigate the effects of hyperglycemia on ubiquitination and endoplasmic reticulum stress in renal intrinsic cells (podocytes and proximal tubular epithelial cells)and its role in pathogenesis of diabetic nephropathy.Methods Diabetic mice were induced by streptozotocin injection.After 16 weeks of hyperglycemia,immunofluorescence was used to detect the expressions of ubiquitination and glucose-regulating protein 94 (GRP94) in renal cortex and medulla area of kidney sections.Primary mouse podocyte and proximal tubular epithelial cells were isolated by flow cytometry,and exposed to 30 mmol/L glucose for indicated time (1 d,3 d and 7 d).Their ubiquitination and GRP94 expressions were evaluated by Western blotting.Results Diabetic mice presented microalbuminuria and slightly widened mesangium was found in glomerular area.Ubiquitinated proteins,mainly localized in podocytes and tubular epithelial cells,exhibited an apparently higher expression in diabetic mice than control mice (all P ＜ 0.05).Hyperglycemia promoted the ubiquitination in a time-dependent manner.Compared with their normal cells,primary mouse podocyte and primary tubular epithilial cells treated with high glucose for 3 d and 7 d showed increased ubiquitinated protein (all P ＜ 0.05).GRP94 was interspersed in podocytes and proximal tubular epithelial cells.Expression of GRP94 was significantly increased in glomerular area of diabetic mice and podocyte with 3 and 7 day-high glucose as compared with those in their control groups (all P ＜ 0.05).GRP94 expression had no significant change in tubular area and tubular epithilial cells treated with high glucose.Conclusions Hyperglycemia may lead to accumulation of ubiquitinated proteins in intrinsic kidney cells.The imbalance of protein homeostasis in podocyte may contribute to podocyte injury during the onset of diabetic nephropathy.