ObjectiveTo prepare triptolide-Chuanxiong Rhizoma extract ethanol transfersomes（TP-CX@TESs）， conduct its quality evaluation， and investigate its in vitro anti-inflammatory efficacy and the underlying mechanisms. MethodsTP-CX@TESs was prepared via the ultrasonic injection method. With encapsulation efficiency and particle size as evaluation indicators， Box-Behnken design-response surface methodology（BBD-RSM） was employed to optimize the formulation process. The TP-CX@TESs prepared under the optimal process was characterized and evaluated for in vitro transdermal performance. A lipopolysaccharide（LPS）-induced RAW264.7 cell inflammation model was established. After 24 h of drug intervention， the levels of inflammatory factors such as nitric oxide（NO）， interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） in the cell supernatant were detected. Western blot was used to determine the protein expression levels of Janus kinase 2（JAK2）， signal transducer and activator of transcription 3（STAT3）， and α7 nicotinic acetylcholine receptor（α7nAChR）， and real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was applied to measure the mRNA expression levels of JAK2， STAT3， the encoding gene of α7nAChR（CHRNA7）， and nuclear transcription factor-κB（NF-κB）. ResultsResults of BBD-RSM showed that the optimal formulation for preparing TP-CX@TESs was as follows：egg yolk lecithin content of 2.3%， ethanol volume fraction of 30%， and ratio of polysorbate-80 to egg yolk lecithin of 2∶5. Microscopic characterization revealed that TP-CX@TESs exhibited a spherical-like structure with a particle size of （105.60±3.85） nm， a polydispersity index of 0.19±0.03， and a Zeta potential of （-15.89±0.98） mV. The encapsulation efficiencies of triptolide， ferulic acid， and ligustilide were （76.88±4.40）%， （78.84±4.40）%， and （65.88±0.06）%， respectively. Both in vitro release and transdermal penetration of triptolide， ferulic acid， and ligustilide in TP-CX@TESs all followed the first-order kinetic model， showing a certain sustained-release property. Experimental results in RAW264.7 cells indicated that TP-CX@TESs significantly inhibited the release of NO， TNF-α， and IL-6（P<0.01）， remarkably upregulated the protein expression levels of STAT3 and α7nAChR（P<0.01）， increased the mRNA expression level of CHRNA7， and significantly downregulated the mRNA expression level of NF-κB（P<0.05， P<0.01）. ConclusionThe optimized formulation process of TP-CX@TESs is simple and feasible， along with favorable in vitro release property， good transdermal permeability， and excellent in vitro anti-inflammatory activity， the mechanism is related to the inhibition of NF-κB.

ObjectiveTo prepare triptolide-Chuanxiong Rhizoma extract ethanol transfersomes（TP-CX@TESs）， conduct its quality evaluation， and investigate its in vitro anti-inflammatory efficacy and the underlying mechanisms. MethodsTP-CX@TESs was prepared via the ultrasonic injection method. With encapsulation efficiency and particle size as evaluation indicators， Box-Behnken design-response surface methodology（BBD-RSM） was employed to optimize the formulation process. The TP-CX@TESs prepared under the optimal process was characterized and evaluated for in vitro transdermal performance. A lipopolysaccharide（LPS）-induced RAW264.7 cell inflammation model was established. After 24 h of drug intervention， the levels of inflammatory factors such as nitric oxide（NO）， interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） in the cell supernatant were detected. Western blot was used to determine the protein expression levels of Janus kinase 2（JAK2）， signal transducer and activator of transcription 3（STAT3）， and α7 nicotinic acetylcholine receptor（α7nAChR）， and real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was applied to measure the mRNA expression levels of JAK2， STAT3， the encoding gene of α7nAChR（CHRNA7）， and nuclear transcription factor-κB（NF-κB）. ResultsResults of BBD-RSM showed that the optimal formulation for preparing TP-CX@TESs was as follows：egg yolk lecithin content of 2.3%， ethanol volume fraction of 30%， and ratio of polysorbate-80 to egg yolk lecithin of 2∶5. Microscopic characterization revealed that TP-CX@TESs exhibited a spherical-like structure with a particle size of （105.60±3.85） nm， a polydispersity index of 0.19±0.03， and a Zeta potential of （-15.89±0.98） mV. The encapsulation efficiencies of triptolide， ferulic acid， and ligustilide were （76.88±4.40）%， （78.84±4.40）%， and （65.88±0.06）%， respectively. Both in vitro release and transdermal penetration of triptolide， ferulic acid， and ligustilide in TP-CX@TESs all followed the first-order kinetic model， showing a certain sustained-release property. Experimental results in RAW264.7 cells indicated that TP-CX@TESs significantly inhibited the release of NO， TNF-α， and IL-6（P<0.01）， remarkably upregulated the protein expression levels of STAT3 and α7nAChR（P<0.01）， increased the mRNA expression level of CHRNA7， and significantly downregulated the mRNA expression level of NF-κB（P<0.05， P<0.01）. ConclusionThe optimized formulation process of TP-CX@TESs is simple and feasible， along with favorable in vitro release property， good transdermal permeability， and excellent in vitro anti-inflammatory activity， the mechanism is related to the inhibition of NF-κB.

Objective To explore the relationship between hemoglobin (Hb) level during the first trimester of pregnancy and gestational diabetes mellitus (GDM). Methods A total of 1 276 participants, who underwent scheduled prenatal examination and normal singleton delivery at the Fifth People′s Hospital of Shanghai and Hospital of Intergrated Chinese and Western Medicine in Minhang District, from January 2016 to May 2018 were included. There were 99 cases of GDM (GDM group) and 1 177 cases of normal (control group) pregnant women.Based on the serum Hb level during the first trimester of pregnancy, participants were divided into three groups, 236 cases of low Hb level group (Hb<110 g/L), 868 cases of normal Hb level group (110 g/L≤Hb<130 g/L), and 172 cases of high Hb level group (Hb≥130 g/L). Maternal clinical data were collected, including Hb level during the first trimester of pregnancy, three-point blood glucose (BG) of oral glucose tolerance test (OGTT) and fasting insulin during the second trimester of pregnancy. Homeostasis model assessment of insulin resistance index (HOMA-IR) and homeostasis model assessment of pancreatic β cell function index (HOMA-β) were used to evaluate insulin resistance and pancreatic β cell function. Results (1) Hb level during the first trimester of pregnancy in GDM group was significantly higher than that in control group [(123±10),(119±11) g/L, P<0.05]. There were no significant difference in gravidity, parity, index of liver and renal function (all P>0.05). (2) Pre-pregnancy body mass index (BMI), 1-hour BG and 2-hour BG of OGTT were significantly increased in the high Hb level group during the first trimester of pregnancy, which were (23±4) kg/m2, (7.3±2.0) mmol/L, and (6.5±1.4) mmol/L (P<0.05), respectively. The pre-pregnancy BMI, 1-hour BG and 2-hour BG of the normal or low Hb level group were (22±3) kg/m2, (6.7±1.6) mmol/L, (6.1± 1.2) mmol/L; (22±3) kg/m2, (6.5±1.5) mmol/L, (5.9±1.1) mmol/L, respectively. There were no statistically significant difference in levels of fasting blood glucose, fasting insulin, HOMA-IR and HOMA-β within 3 groups (all P>0.05). (3) In the high Hb level group, prevalence of pregnancy overweight or obesity and GDM were the highest, which were 37.2%(64/172) and 15.1%(26/172), respectively; the differences were statistically significant (all P<0.05). (4) The serum Hb level in the first trimester was positively related with pre-pregnancy BMI (r=0.130, P<0.05), 1-hour BG (r=0.129, P<0.05), 2-hour BG (r=0.134, P<0.05), fasting insulin (r=0.096, P<0.05), and HOMA-IR (r=0.101, P<0.05).Logistic regression indicated that Hb≥130 g/L during the first trimester of pregnancy was an independent risk factor for GDM ( OR=2.799, 95% CI :1.186-6.604; P<0.05). Conclusion The high level of Hb (Hb≥130 g/L) during the first trimester of pregnancy is associated with GDM.

Objective: To explore the relationship between hemoglobin (Hb) level during the first trimester of pregnancy and gestational diabetes mellitus (GDM). Methods: A total of 1 276 participants, who underwent scheduled prenatal examination and normal singleton delivery at the Fifth People's Hospital of Shanghai and Hospital of Intergrated Chinese and Western Medicine in Minhang District, from January 2016 to May 2018 were included. There were 99 cases of GDM (GDM group) and 1 177 cases of normal (control group) pregnant women.Based on the serum Hb level during the first trimester of pregnancy, participants were divided into three groups, 236 cases of low Hb level group (Hb<110 g/L), 868 cases of normal Hb level group (110 g/L≤Hb<130 g/L), and 172 cases of high Hb level group (Hb≥130 g/L). Maternal clinical data were collected, including Hb level during the first trimester of pregnancy, three-point blood glucose (BG) of oral glucose tolerance test (OGTT) and fasting insulin during the second trimester of pregnancy. Homeostasis model assessment of insulin resistance index (HOMA-IR) and homeostasis model assessment of pancreatic β cell function index (HOMA-β) were used to evaluate insulin resistance and pancreatic β cell function. Results: (1) Hb level during the first trimester of pregnancy in GDM group was significantly higher than that in control group [(123±10),(119±11) g/L, P<0.05]. There were no significant difference in gravidity, parity, index of liver and renal function (all P>0.05). (2) Pre-pregnancy body mass index (BMI), 1-hour BG and 2-hour BG of OGTT were significantly increased in the high Hb level group during the first trimester of pregnancy, which were (23±4) kg/m², (7.3±2.0) mmol/L, and (6.5±1.4) mmol/L (P<0.05), respectively. The pre-pregnancy BMI, 1-hour BG and 2-hour BG of the normal or low Hb level group were (22±3) kg/m², (6.7±1.6) mmol/L, (6.1±1.2) mmol/L; (22±3) kg/m², (6.5±1.5) mmol/L, (5.9±1.1) mmol/L, respectively. There were no statistically significant difference in levels of fasting blood glucose, fasting insulin, HOMA-IR and HOMA-β within 3 groups (all P>0.05). (3) In the high Hb level group, prevalence of pregnancy overweight or obesity and GDM were the highest, which were 37.2%(64/172) and 15.1%(26/172), respectively; the differences were statistically significant (all P<0.05). (4) The serum Hb level in the first trimester was positively related with pre-pregnancy BMI (r=0.130, P<0.05), 1-hour BG (r=0.129, P<0.05), 2-hour BG (r=0.134, P<0.05), fasting insulin (r=0.096, P<0.05), and HOMA-IR (r=0.101, P<0.05).Logistic regression indicated that Hb≥130 g/L during the first trimester of pregnancy was an independent risk factor for GDM (OR=2.799, 95%CI: 1.186-6.604; P<0.05). Conclusion The high level of Hb (Hb≥130 g/L) during the first trimester of pregnancy is associated with GDM.

Objective To explore the diagnostic value of magnetic resonance diffusion weighted imaging(DWI),3D time of flight magnetic resonance angiography(3D-TOF MRA),and 3D arterial spin labeled MR perfusion imaging(3D-ASL)in the senile ischemic cerebrovascular diseases.Methods Ninety patients with acute cerebral infarction and 69 with transient ischemic attack were retrospectively analyzed based on data of DWI,3DTOF MRA and 3D-ASL MRI.Additionally,60 healthy individuals were recruited as a control group.SPSS 21.0 statistical software was used.Results In 90 acute cerebral infarction(ACI)group,the 3D-ASL MRI showed hypoperfusion in 85 patients,hyperperfusion in 2 patients,and no apparently perfusion abnormality in 3 patients.In 85 hypoperfusion patients,the average hypoperfusion area showed by 3D-ASL MRI was(2 543±1 133)mm2,which was higher than the area of high signal in DWI(817±413) mm2 (P＜0.01),with ASL＞DWI in 83 cases and ASL≈DWI in 2 cases.In 69 transient ischemic attack patients,25 patients had artery stenosis and abnormal ASL,11 patients had artery stenosis and normal ASL,17 patients had normal vascular and abnormal ASL,15 had normal vascular and normal ASL.The accuracy,sensitivity,specificity,positive predictive value,and negative predictive value were higher in 3D-TOF MRA combined with 3D-ASL than in their individual inspection(x2=10.35,P＜0.01,P＜0.05),and the rates of missed diagnosis and misdiagnosis were lower in 3D-TOF MRA combined with 3D-ASL than in their individual inspection (x2 =4.93,P ＜ 0.05).Conclusions ASL,MRA,and DWI can =reflect the different characteristics of the senile ischemic cerebrovascular diseases,and should be used as the standard sequence in geriatric neurology to provide more comprehensive information for the diagnosis and treatment of senile ischemic cerebrovascular disease.

Objective To investigate the effect of emodin on immune suppression function of regulatory T cells in a mouse model of CT26 colon cancer. Methods Twenty-four mice were divided into the negative control group, the emodin group and the tumor group. The populations of CD8+CD3+T cells, the T cells producing IFN-γand the CD4+CD25+Tregs secreting IL-10 in different mouse tissues were detected by flow cytometry. Levels of IFN-γ, TNF- β1 and IL-10 in serum were determined by ELISA. Results Emodin could significantly increase the percent of CD8+CD3+T cells in tumor (P < 0.05) and improve the ability of IFN-γ secretion in T cells from peripheral blood and lymph nodes (P < 0.05). Emodin could reduce the levels of IFN-γ, TNF-β1 and IL-10 in the serum (P < 0.01) and inhibit IL-10 secretion in CD4+CD25+ Tregs (P < 0.01). Conclusion Emodin possesses the antitumor effect by affecting the immunosuppressive function of Tregs cells.

Objective To detect the immune effect of FbaAmAb2 against the surface protein of group A Straptococcus (GAS),and explore the pathogenesis and therapy of GAS infections.Methods By subclonal and bacterial ELISA,the positive hybridoma cells were screened that can produce better titers of FbaAmAb2 against GAS-surface FbaA protein,and were injected into the peritoneal cavities of BALB/c mice to produce ascites.The collected ascites were performed to dilute,as follows,original ascite,1:2,1:4,1:8,and 1:16 to test tube agglutination.Based on the results,we selected appropriate dilution to passively immunize mice,and then challenged the mice with GAS,evaluating FbaAmAb2 neutralizing ability with GAS in mice by the survival rate of the immunized mice.Whether FbaAmAb2 could inhibit the binding of factor H to GAS was confirmed by the invasive inhibition assay.Results The IgG titer of bacteria solution ELISA is 1:160 and the titer of tube agglutination is 1∶8.The protect rates of FbaAmAb2 on preventing mice with GAS infections are as follows:66.67％ in original ascite and 1:2 diluted groups,and 50％ in 1:4 diluted group.Mice in each experimental group were evoked significantly protective immune responses compared with the PBS control by SPSS analysis.FbaAmAb2 can competitively inhibit factor H binding to the surface proteins FbaA of GAS,which decreased the entry of GAS into the cytoplasm of human epithelial cells through the binding of factor H.Conclusion FbaAmAb2 is promising to be used in emergent prevention or the clinical therapy for GAS infection and it is promising starting points for pharmacologic targeting and further development of new therapeutic agents for GAS.

Objective To investigate immune responses and protective effect induced by two recombinant proteins of hepatitis C virus(HCV)in BALB/c mice.Methods BALB/c mice were immunized with recombinant proteins HCV-T and(or)HCV-F4HVR1 three times.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Five mice were sacrificed after 14 days of the last immunization.Splenic cells were isolated and levels of interferon(IFN)-γ,interleukin(IL)-4 and cytotoxic T lymphocyte(CTL)cytotoxicity assay were measured in vitro.The remaining mice were subcutaneously injected with 1.0×106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results Compared with phosphate bufter saline(PBS)group,combined immunization with HCV-T and HCV-F4HVR1 induced higher levels of specific IgG against HCV-F4HVR1(t=3.815,3.762,P＜0.05),HCV-NS3-specific CTL response(t=3.971,P＜0.05)and IL-4(t=3.512,3.417,P＜0.05)and IFN-γ(t=3.813,3.426,3.671,P＜0.05)secretions.Conclusion High levels of specific humoral immunity and cellular immunity are induced in vivo after combined immunization with HCV-T and HCV-F4 HVR1,which could effectively prevent from the attack of SP2/0-NS3 cells.

Objective To investigate the cellular and humoral immune responses and protective effect induced by co-immunization with two multi-epitope combinant antigens. Methods Mice were co-im-munized with the muhi-epitope HCV-T and HCV-E1 antigens three times. Sera antibodies IgG, IgG1 and IgG2a were tested by ELISA. Spleens from BALB/c mice immunized were removed 10 days after the last im-munization. CTL activity was assessed using LDH cytotoxicity assay kit. IFN-γ- and IL-4-secreting cells were quantified using ELISPOT kit. Two weeks after the final immunization, the mice were challenged sub-cutaneously(s, c. ) at the back with 106 SP2/0-NS3 cells, and protective effect was observed. For therapy, 106 SP2/0-NS3 cells were implanted into the back of BALB/c mice. Seven days later, mice were immuniza-tion three times. Therapy effect was observed. Results Co-immunization with HCV-T and HCV-E1 induced high tiers of HCV-El-specific IgG, IgG1 and IgG2a antibodies, and high level of CTL activity. Synergistic effect in frequencies of both specific IFN-γ-secreting cells and IL-4-secreting cells was observed in mice co-immunized. Prophylactic as well as therapeutic administration of mT + mE1 in mice led to protecting mice against SP2/0-NS3 cells. These results suggested that mT + mE1 was potential as a prophylactic as well as therapeutic HCV vaccine. Conclusion Co-immunization with HCV-T + HCV-EI induced protective humor-al and cellular immune response. HCV-T + HCV-E1 was potential as a recombinant HCV vaccine.

BACKGROUND: Whether horizontal and vertical impacts cause craniocerebral injury of the same degree, and whether serum myelin basic protein (MBP) changes is associated with the direction of impact have been scarcely addressed in previous studies.OBJECTIVE: To observe the change in the serum MBP levels and morphological changes of the brain tissue rabbits with craniocerebral injury resulting from impacts from different directions.DESIGN: A randomized controlled experiment.SETTING: Department of Neurosurgery of a hospital affiliated to a medical university.MATERIALS: This study was conducted in the Central Laboratory of Shenyang Medical College between October 2003 and January 2004. Totally 30 healthy rabbits of either sex were randomly divided into two equal groups to receive vertical and horizontal impact on the head.METHODS: All the rabbits were anesthetized and fixed in prone position onto a table equipped with a device for inducing impact on the head from different directions. Venous blood of 1 mL was taken from the edge of the ear of each rabbit for detecting MBP before injury. The rabbits in vertical impact group received the impact of a hammer falling directly on the parietal bone of the skull. In the horizontal group, the rabbits were subjected to horizontal impact on the lateral side of the skull. Forty-eight hours after the injury, venous blood were again taken for MBP measurement. The rabbits were then killed for pathological examination of the brain.MAIN OUTCOME MEASURES: Changes in serum MBP and pathomorphology of the brain tissues in the two groups after the impact.RESULTS: ① According to intention to treat analysis, all the 30 animals were involved in the experiment. There was no significant difference in serum MBP levels between the two groups before the impact, nor 48 hours after the impact( P ＞ 0.05). In the vertical impact group, serum MBP levels before the impact was( 1.68 ± 0. 86) μg/L, which was significantly lower than that after the impact[ (5.25 ± 1.96) μg/L, t = 3. 226, P ＜ 0. 05]. ② In the horizontal strike group, serum MBP also significantly increased from the level of( 1.70 ± 0.91 ) μg/L before impact to(5.73 ± 2.07) μg/L after that( t = 3. 080, P ＜ 0.05) . ③ After the impact, vasodilation and congestion in the cortex near the midline of the bilateral parietal region and edema in the deep layer of the white matter were observed in the vertical impact group, along with significant enlargement of the extravascular and extracellular space. In the horizontal group, similar changes except those near the midline of the bilateral parietal region took place after the impact; the same pathological changes were also observed in the basal plane, with nearly identical pathological changes in the deep layer of the white matter.CONCLUSION: Vertical and horizontal impacts can both result in almost identical brain injuries demonstrated by similar changes in serum MBP and the pathomorphology of the brain tissues after the impacts.