Objective To evaluate the effects of tacrolimus on the secretion of IL-6 and sIL-2R as well as the expression of IL-6 and sIL-2R mRNA by lymphocytes.Methods Jurkat human lymphoma cells were cultured and treated with tacrolimus of different concentrations.Enzyme linked immunosorbent assay was performed to determine the levels of IL-6 and sIL-2R in the supernatant of Jurkat cells at 48 hours after treatment with tacrolimus of 0,10,102,103 and 104 nmol/L,and real time reverse transcription PCR to measure the expression of IL-6 mRNA and sIL-2R mRNA of Jurkat cells at 48 hours after treatment with tacrolimus of 102 nmol/L.Results Tacrolimus of 102 - 104 nmol/L could suppress the secretion of IL-6 and sIL-2R from Jurkat cells (all P＜ 0.05),with a more marked suppressing effect achieved by the use of tacrolimus at 103 - 104 nmol/L.The expressions of IL-6 and sIL-2R mRNA from Jurkat cells were downregulated by tacrolimus of 102 nmol/L (both P ＜ 0.05).Conclusion Tacrolimus at certain concentrations could downregulate the secretion of IL-6 and sIL-2R as well as the expression of IL-6 and sIL-2R mRNA by lymphocytes.

Objective To investigate the expression of apelin and its recoptor (APJ) in myocardium in insulin-resistant CIR rats with myocardial ischemia.Methods Totally 24 male SD rats were randomly divided into three groups:IR group,IR+ischemia group,the control group (n=8 each).Rats in IR and IR+ischemia groups were fed with the high fat diet.Rats in control group were given the basic diet.The rat model of insulin resistance was assessed by fasting blood glucose (FBG),fasting insulin (Fins) and insulin resistance index (HOMA-IR).The rat model of myocardial ischemia was conducted by subcutaneous injection of isoprenaline 1 mg/kg per day in IR+ ischemia group.The other groups were injected an equal volume of saline.The expression levels of apelin and APJ mRNA in myocardium were determined by real-time-PCR.The positive expression of apelin polypeptide was detected by immunochemistry.Results Compared with the control group,the apelin average optical density was increased in IR group and was decreased in IR+ ischemia group [(0.16±0.004) vs.(0.13±0.005),(0.10±0.002) vs.(0.13±0.0050),both P＜0.05].There was a significant difference in apelin average optical density between IR group and IR+ischemia group [(0.16± 0.004) vs.(0.10±0.002),P＜0.05].Compared with the control group,the relative expression of apelin mRNA was increased in IR group [(5.89±0.36) vs.(4.40±0.24),P＜0.05]The relative expression of apelin mRNA was decreased in IR± ischemia group as compared with IR group [(2.66 ± 0.17) vs.(5.89 ± 0.36),P ＜ 0.05].The APJ mRNA relative expression was increased in IR group as compared with control group and was decreased in IR+ ischemia group as compared with IR group [(10.46±1.06) vs.(6.54±0.63),(3.31±0.31) vs.(10.46±1.06),both P＜0.05].Conclusions The expressions of apelin and APJ are inhibited in insulin-resistant rats with myocardial ischemia,which attenuates their protective effects on myocardium.

Objective: To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved, specifically focusing on the role of the caspase-3/gasdermin E (GSDME) signaling pathway and the impact of endoplasmic reticulum (ER) stress and autophagy. Methods: Necrostatin-1 (Nec-1), lactate dehydrogenase release (LDH) assay, and Hoechst/propidium iodide (PI) double staining were employed to validate the mode of cell death. Western blot was used to detect the cleavage of GSDME and the expression of light chain 3 (LC3) and BIP. Results: Celastrol induced cell swelling with large bubbles, which is consistent with the pyroptotic phenotype. Moreover, treatment with celastrol induced GSDME cleavage, indicating the activation of GSDME-mediated pyroptosis. GSDME knockout via CRISPR/Cas9 blocked the pyroptotic morphology of celastrol in HeLa cells. In addition, cleavage of GSDME was attenuated by a specific caspase-3 inhibitor in celastrol-treated cells, suggesting that GSDME activation was induced by caspase-3. Mechanistically, celastrol induced endoplasmic reticulum (ER) stress and autophagy in HeLa cells, and other ER stress inducers produced effects consistent with those of celastrol. Conclusion These findings suggest that celastrol triggers caspase-3/GSDME-dependent pyroptosis via activation of ER stress, which may shed light on the potential antitumor clinical applications of celastrol.

OBJECTIVE:To prepare Puerarin polymeric micelles and establish a method to determine its entrapment efficiency. METHODS:Puerarin polymeric micelles were prepared by film dispersion method. The polymeric micelles and free drug were sepa-rated by centrifugal-millipore filter filtration method. The entrapment efficiency of puerarin polymeric micelles was determined by HPLC. Diamonsil C18(2)column was used with 1% citric acid solution-methanol(65∶35)at the flow rate of 1 ml/min. The detec-tion wavelength was set at 250 nm,and column temperature was room temperature. RESULTS:The prepared polymeric micelles were spherical and spherical-like in shape with a mean particle size of 54.12 nm,polydispersity index of 0.122,Zeta potential of -13.60 mV;the linear range of puerarin was 2-10μg/ml(R2=0.999 4)with average recovery rate of 99.2%(RSD=0.9%,n=3). The re-covery rate of free drug was 95.3%(RSD=1.7%,n=3). The mean entrapment efficiency and drug-loading amount of puerarin were(35.5±2.12)% and(0.3±0.07)%,respectively(n=3). CONCLUSIONS:Film dispersion method is suitable for the prepara-tion of Puerarin polymeric micelles. Established method is convenient,accurate and reliable for the content and entrapment efficien-cy determination of Puerarin polymeric micelles.

Objective To compare the effects of selenium nanoparticles (Nano-Se) and sodium selenium (Na2SeO3) on apoptosis and reactive oxygen species (ROS) of articular chondrocytes from patients with Kashin-Beck Disease (KBD) in vitro,and provide a scientific basis for preventing KBD.Methods The subjects with KBD were diagnosed on National Clinical Diagnostic Criteria of KBD (WS/T207-2010),articular cartilage from 8 patients undertaken joint replacement operation were collected.In vitro,chondrocytes were treated with concentration of 0,25,50,100,200,300,400 and 500 μg/L of Nano-Se and Na2SeO3 for 5 d,respectively.Cell growth was detected by MTT assay,and the highest concentration and time corresponding to the highest survival rate of Nano-Se and Na2SeO3 were used in the following experiment.KBD chondrocytes were treated with Nano-Se and Na2SeO3,and divided into control group,Na2SeO3 group,Nano-Se group according to the randomized design.Each group had 8 cases.The cell apoptosis and ROS were detected by flow cytometry.Results The optimal intervention concentration of Nano-Se and Na2SeO3 was 100 and 400 μg/L,respectively.The optimal intervention time of NanoSe and Na2SeO3 both was 3 days.There was a significant decrease in the total and terminal apoptosis,ROS level of chondrocytes in Nano-Se group [(4.67 ± 0.89)％,(1.51 ± 0.48)％,(56.04 ± 4.81)％] and Na2SeO3 group [(7.07 ±0.25)％,(4.37 ± 0.37)％,(87.13 ± 6.60)％] compared with those of control group [(9.95 ± 0.38)％,(6.93 ± 0.42)％,(125.17 ± 16.60)％,all P ＜ 0.01].The difference of early apoptotic rate among control group,Na2SeO3 group,NanoSe group [(3.02 ± 0.41)％,(2.7 ± 0.46)％,(3.16 ± 0.56)％] was not statistically significant (F =2.11,P =0.35).Conclusion Appropriate concentration of Nano-Se can significantly decrease oxidative stress of KBD chondrocytes and inhibit apoptosis compared to Na2SeO3.

Objective To evaluate the role of C-type lectin domain family 2,member B (CLEC2B) gene in the pathogenesis of vitiligo.Methods Real time fluorescence-based PCR was performed to detect the expression of CLEC2B mRNA in the peripheral blood and lesional skin of 37 patients with vitiligo as well as in the peripheral blood and normal skin of 40 healthy controls.Data were statistically analyzed by t test and chisquare test.Results Among the 37 patients,23 had progressive vitiligo,14 stable vitiligo,31 vitiligo vulgaris,6 segmental vitiligo.The expression level of CLEC2B mRNA was significantly higher in vitiligo lesions than in the control skin (1.21 ± 0.03 vs.1.00,t =4.432,P ＜ 0.05),but was of no significant difference in peripheral blood between the patients and healthy controls (1.02 ± 0.05 vs.1.00,t =1.435,P ＞ 0.05).Increased expression of CLEC2B mRNA was noted in lesions of vulgaris vitiligo compared with those of segmental vitiligo (1.21 ± 0.03 vs.1.02 ± 0.01,t =5.330,P ＜ 0.05),as well as in lesions of progressive vitiligo compared with those of stable vitiligo (1.25 ± 0.05 vs.1.08 ± 0.03,t =3.046,P ＜ 0.05).No significant difference was observed in the expression of CLEC2B mRNA among lesions of vitiligo with different courses (P ＞ 0.05).Conclusion The differential expression of CLEC2B mRNA may take part in the pathogenesis of vitiligo.

OBJECTIVE： To prepare Paclitaxel（PTX）nanostructured lipid carriers （NLC） modified by small peptide alanine-glutamic acid-tyrosine-leucine-arginine （AEYLR）， and to evaluate its anti-tumor effect in vitro and in vivo. METHODS： NLC， PTX-NLC （P-NLC） and AEYLR modified P-NLC （A-P-NLC） were prepared by emulsion evaporation-low temperature solidification curing method. Its appearance， particle size， multi-dispersion index（PDI） and Zeta potential were characterized，encapsulation rate，drug loading and in vitro drug release were detected respectively. Using NCI-H1299 and S180 cells as objects， CCK-8 method was adopted to investigate inhibitory effects of free PTX， P-NLC and A-P-NLC （0.44-44.00 μg/mL， by PTX） to those cells. The half inhibition concentration （IC50） was calculated. Using S180 tumor-bearing mice as model animal， anti-tumor effects of free PTX， P-NLC and A-P-NLC （5 mg/kg， by PTX） were evaluated. RESULTS： P-NLC and A-P-NLC were round-like and dispersed evenly. The particle size， PDI and Zeta potential of A-P-NLC were （43.92±0.76） nm， 0.203±0.034 and （－19.77±1.16） mV， which were all increased to certain extent， compared with P-NLC. The encapsulation efficiency and drug loading of A-P-NLC were （95.71±0.68）％ and（1.97±0.25）％， which were both decreased to certain extent， compared with P-NLC. The cumulative release rate of A-P-NLC was（35.17±2.08）％ within 48 h， showing significant sustained-release effect compared with free PTX； the release of A-P-NLC was slower than P-NLC. Compared with free PTX and P-NLC， inhibitory rates of same concentration of A-P-NLC to NCI-H1299 cells and S180 cells were almost increased significantly， while IC50 values were all decreased significantly. There was no death in S180 tumor-bearing mice treated with A-P-NLC and the general condition was good； the volume of tumors was significantly reduced， the mass of tumors was significantly reduced， and the inhibition rate of tumors was significantly increased （P＜0.05 or P＜0.01）. CONCLUSIONS： A-P-NLC has significantly sustained-release effects； its inhibitory rate to NCI-H1299 cells and S180 cells in vitro， and its inhibitory effects on S180 solid tumor in mice are all better than free PTX and P-NLC， while the toxicity is decreased to certain extent.