Objective:To investigate the role of dopamine receptors in the central amygdala (CeA) in reduction of the anxiety level by propofol in a mouse model of post-traumatic stress disorder (PTSD).Methods:Fifty-six SPF healthy adult male C57BL/6 mice, aged 10 weeks, weighing 20-25 g, were divided into 7 groups ( n=8 each) using a random number table method: control group (C group), PTSD group (P group), PTSD+ propofol group (PP group), PTSD+ fat emulsion group (PF group), PTSD+ propofol+ normal saline group (PPN group), PTSD+ propofol+ dopamine receptor D1 (DRD1) antagonist group (PP+ DRD1-Ant group), and PTSD+ propofol+ DRD2 antagonist group (PP+ DRD2-Ant group). The PTSD model was developed by continuous plantar electric shock for 3 days. Propofol 120 mg/kg was intraperitoneally injected after successful establishment of the model in PP group, and the equal volume of fat emulsion was intraperitoneally injected in PF group. In PPN group, PP+ DRD1-Ant group and PP+ DRD2-Ant group, the equal volume of normal saline, DRD1 antagonist hydrochloride and DRD2 antagonist eticlopride hydrochloride were injected in bilateral CeA regions, respectively, 30 min later the efficacy of drugs reached the peak, and then propofol 120 mg/kg was intraperitoneally injected. The anxiety levels were measured at 4 h (T 1) and day 3 after propofol injection (T 2) by the open field test and elevated cross maze test. Results:Compared with C group, the time spent entering the open and central areas was significantly shortened at T 1, 2, and the number of entering the open and central areas was decreased at T 1, 2 in P group ( P<0.001). Compared with P group, the time spent entering the open and central areas was significantly prolonged at T 1, the number of entering the open and central areas was increased at T 1 ( P<0.001), and no significant change was found at T 2 in PP group ( P>0.05), and no significant change was found in the aforementioned parameters at T 1, 2 in PF group ( P>0.05). Compared with PPN group, the time spent entering the open and central areas was significantly shortened at T 1, and the number of entering the open and central areas was decreased at T 1 in PP+ DRD2-Ant group ( P<0.001), and no significant change was found at T 1 in PP+ DRD1-Ant group ( P>0.05). Conclusions:Activation of DRD2 in the CeA is involved in the process by which propofol reduces the anxiety level of mice with PTSD.

Purpose Based on European lumbar spine phantom,to investigate the effect of different noise index(NI)combined with adaptive statistical interactive reconstruction-veo(ASiR-V)weights on the measurement of lumbar spine bone density,and to explore the optimal combination of the two parameters.Materials and Methods Using GE Revolution CT spectral imaging scanning,54 groups of scanning parameters with NI values of 4-20(interval 2)combined with ASiR-V weights of 0-100%(interval 20%)were selected for spectral scanning of European lumbar spine phantom.Regions of interest were placed in the middle of L1,L2 and L3,respectively.hydroxyapatite(HAP)-H2O based substances were selected to measure the HAP content of each vertebral body.The differences between the measured bone density value and the true value under different NI combined with different ASiR-V were compared to evaluate its accuracy.Results There were statistically significant differences in HAP measurements of L1,L2 and L3 vertebra in 54 groups of scanning conditions(all P<0.001).When NI=14,ASiR-V80%;NI=16,ASiR-V100%;NI=18,ASiR-V60%;NI=18,ASiR-V80%;NI=20,ASiR-V60%;NI=20,ASiR-V80%;NI=20,ASiR-V100%,there was no statistically significant difference between HAP measured value and true body model value(P>0.05).Conclusion With NI=18,ASiR-V60%,spectral CT can accurately measure lumbar spine bone density and significantly reduce the radiation dose.In clinical application,bone density can be measured by low dose scanning by increasing the weight of NI and ASiR-V.

Objective Network pharmacology and molecular docking and molecular dynamics techniques were used to investigate the mechanism of action of Alhagi sparsifolia Shap.in the treatment of sepsis and to perform animal experimental verification.Methods First,we screened the effective ingredients and their action targets of Alhagi sparsifolia Shap.,meanwhile,screened relevant action targets for the treatment of sepsis,constructed a protein interaction(PPI)network,and performed topology analysis to draw a TCM disease target network diagram.Second,Kyoto Encyclopedia of genes and genomes enrichment analysis was performed for core targets in the network diagram,along with gene ontology functional enrichment analysis.This was followed by molecular docking and molecular dynamics simulation experiment validation of the core targets.Finally,mice were used for the verification of animal experiments.Results Thirty active components of Alhagi sparsifolia Shap.were screened out,and the top 5 ranked by degree value were quercetin,(-)-epigallocatechin,(-)-Epigallocatechin Gallate,genistein,kaempferol and epigallocatechin with 196 action targets;2144 disease-related targets for sepsis,105 targets for Alhagi sparsifolia Shap.-sepsis intersection,and the core targets were TNF,IL-6,AKT1,VEGFA,CASP3,IL-1β Et al.PI3K-Akt,TNF,HIF-1,AGE-RAGE,IL-17 and other signaling pathways are involved to mediate inflammatory responses,apoptosis and other biological processes to exert therapeutic effects on sepsis.Molecular docking results showed that camelina flavanoids bound equally well to each key target,among which the conformations with the lowest binding energy were(-)-Epigallocatechin Gallate-IL-6 and quercetin-IL-6.Molecular dynamics simulations were performed on the two pairs of complexes,and the results indicated that the stable binding could be achieved through a combination of electrostatic,van der Waals potential,and hydrogen bonding interactions.Animal experiments confirmed that Alhagi sparsifolia Shap.could inhibit the activation of PI3K/Akt signaling pathway,decrease the protein expression of Caspase-3,VEGF and reduced peripheral blood inflammatory factors secretion of TNF-α、IL-1βand IL-6,alleviating inflammatory injury in tissues and organs.Conclusion The therapeutic effect of Alhagi sparsifolia Shap.on sepsis is achieved through multi biological processes,multi targets,and multi pathways.It provides a certain theoretical basis for the clinical application of camel spines as well as sepsis treatment.

Objective: To investigate the expression pattern，underlying function and clinical significance of Guanylate-binding protein 1 ( GBP1) in pulmonary tuberculosis ( pTB) ． Methods: Immunohistochemical staining was applied to detect the expression of GBP1 in pTB specimensand control samples． Combined with Gene Expression Omnibus ( GEO) datasets ，including GSE83456 and GSE34608，receiver operating characteristic ( ROC) curve was depicted to assess the diagnostic value of GBP1 in pTB．Then，the correlation between GBP1 and related regulatory factors was analyzed by protein-protein interaction network ( PPI) ; Finally，the potential molecular mechanism of GBP1 in pTB was probed by Gene Set Enrichment Analysis( GSEA) . Results: Compared with the control group，GBP1 was significantly overexpressed in human pTB samples，including lung tissue and blood．The positive rate of GBP1 protein in pTB was 73. 9% . ROC curve analysis revealed that GBP1 might have important value in early diagnosis of pTB．GSEA analysis suggested that the hyper-expression of GBP1 was closely related to the host inflammatory response，IFN-γ/ α signaling pathway and TNF-α/ IL-6 signal transduction． Conclusion GBP1 is highly expressed in pTB tissues and is involved in the process of inflammatory response and host anti-tuberculosis infection ; GBP1 may be used as an early diagnostic marker or therapeutic target for pTB.

Spinal cord injury has a high rate of disability in clinical practice, which can be divided into complete SCI and incomplete SCI according to different injury segments and severity.The main purpose of treatment is to protect the nerves.At present, acute spinal cord injury is mainly treated with surgical decompression, neurotrophic treatment, hormone therapy, hypothermia therapy, rehabilitation intervention and other clinical comprehensive treatment.In recent years, breakthroughs have been made in the field of endogenous and exogenous neural stem cell research, and important progress has been made in the basic research of stem cell transplantation.In the long run, nerve regeneration and nerve modulation may be the most promising therapy for the repair of spinal cord injury.

We studied the effect of the Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-proteasome system on mono-resistant to rifampin resistance to M.tuberculosis.A resazurin-based assay was employed to evaluate minimum inhibitory concentration (MIC) and comparative research on mono-resistant to rifampin MTB with Pup,Dop,PafA,Mpa genes expression and deletion of the difference.Above testing strains,respectively,carbonyl cyanide chlorobenzene hydrazone (CCCP),reserpine (RP),verapamil (VP)and chlorpromazine (CPZ) were tested.We compared and analyzed the change of rifampicin MICs on the various strains.Compared with rifampin resistant MTB,overexpression of Pup,Dop,PafA and Mpa genes were able to make monorifampicin of M.tuberculosis to enhance resistance to rifampin.Deletion of Pup gene,Mpa gene,Dop gene,PafA gene significantly decreased the resistance to rifampicin alone MTB,and the P value was ＜0.05.Results indicated that 4 kinds of efflux pump inhibitors can reduce the degree of rifampin MIC in different strains.Through the factorial analysis,there were some interactions between MTB and PPS efflux pump inhibitors,and the P value was ＜0.05.MTB PPS has influence on mono-rifampin resistance to MTB and it may regulate the efflux pathway related protein to influence its resistance.

Objective To study the expressions of myeloid cell leukemia-1 (Mcl-1 ) gene in mouse macrophages Raw264.7 and human macrophages THP-1,to screen out the cell lines with high levels of expression as the experimental cells,and based on the screening results to construct the short hairpin RNA(shRNA)eukaryotic expression plasmid targeting mice Mcl-1 gene for transfection and further screen out the shRNA expression plasmid with the most obvious effect in silencing Mcl-1 gene.Methods Semi-quantitative PCR method was used to detect the expression of Mcl-1 mRNA in the two kinds of macrophages.Western blotting was adopted to detect the expressions of Mcl-1 proteins in the two kinds of macrophages.Three different gene loci for Mcl-1 shRNA fragments were designed with small molecules interfering RNA (siRNA)software.Eukaryotic expression plasmid Mcl-1 shRNA 1-3 carrying the shRNA fragments was constructed by a company.And the eukaryotic expression plasmid vector was transfected into scavenger cells Raw264.7 mice via through the liposome.The transfection results 24 h and 48 h after transfection were observed under the inverted fluorescence microscope;Mcl-1 mRNA and protein expression were detected by real time quantitative PCR and Western blot,respectively.Results The relative expression levels of Mcl-1 mRNA and protein in mouse macrophages Raw264.7 were significantly higher than those of human macrophages (P <0.05).The shRNA expression vector constructed within 24 h and 48 h could decrease Mcl-1 mRNA and protein levels in Raw264.7 cells,especially with the most obvious silencing effect at 48 h.The 48-h transfection group differed significantly from normal group,liposome group and negative control group (P <0.05).Compared with Mcl-1shRNA1and Mcl-1shRNA2,Mcl-1shRNA3 had the strongest effect in silencing Mcl-1mRNA and protein.Conclusion We have successfully screened out experimental Raw264.7 cells and Mcl-1 shRNA eukaryotic expression plasmid which has an obvious silencing effect targeting on Mcl-1 in mice macrophages Raw264.7.

[ ABSTRACT] AIM: To explore the effects of Mcl-1 signal pathway blockers on Mcl-1 expression, macrophage apoptosis and Mycobacterium tuberculosis in the model of mice infected with Mycobacterium tuberculosis H37Rv.METH-ODS:A mouse infection model was established by intraperitoneal injection of H37Rv suspension.The signaling pathway blockers AG490, PD98059 and LY294002 for JAK/STAT, MAPK and PI3K, respectively, were intraperitoneally injected into the mice infected with H37Rv.Cell acid-fast staining was used to observe whether the mouse peritoneal macrophages infected with H37Rv were successfully established.Immunocytochemical method was employed to detect Mcl-1 expression in the mouse peritoneal macrophages infected with H37Rv.The apoptotic rate in each group was measured by flow cytomer-ty.The scavenging capacity of apoptotic macrophages against H37Rv was determined by Mycobacterium tuberculosis colony counting.RESULTS:The result of cell acid-fast staining revealed the existence of dispersive arrangement of red short anti-acid Mycobacterium tuberculosis within infected macrophages.The result of cell immunocytochemistry showed strongly posi-tive expression of Mcl-1 protein in H37Rv infection group, AG490 treatment group and LY294002 treatment group, weakly positive expression of Mcl-1 protein in PD98059 treatment group, and negative expression of Mcl-1 protein in control group. The result of flow cytometry found that the macrophage apoptotic rate in H37Rv infection group was higher than that in con-trol group, while that in PD98059 treatment group was high than that in other groups with statistically significant differences (P<0.05).The result of Mycobacterium tuberculosis colony counting showed that PD98059 treatment had the most signifi-cant inhibitory effect on H37Rv strain.CONCLUSION: Mcl-1 signaling pathway blockers increase the apoptotic rate of macrophages infected with Mycobacterium tuberculosis H37Rv and inhibit the growth of Mycobacterium tuberculosis by inhibi-ting the signaling pathways of JAK/STAT, MAPK and PI3K, among which the MAPK has the most obvious interfering effect on Mcl-1, and leads to the highest apoptotic rate of infected macrophages and the strongest bacteriostasis.

AIM:To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference .METH-ODS:The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang , H37Rv, H37Ra and BCG.Mcl-1-shRNA was applied to the mouse model of infection , and the control groups were set up .On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected .The expression of Mcl-1 at mRNA and protein levels was deter-mined by real-time PCR and Western blot .The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry . RESULTS:The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05).The expres-sion of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group ( P<0.05 ) . Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice .CONCLUSION: The Mcl-1 ex-pression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tu-berculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis .

Objective:To transfect Mcl-1shRNA into Raw264.7 cells,and screen out specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene to figure out the effect of shRNA on Mcl-1 expression in murine macrophage cell line Raw 264.7.Methods: Specific shRNA was transfected into murine macrophage cell line Raw 264.7 via lipofectamine.Semi-quantitative RT-PCR and Western blot were respectively employed to test the changes in Mcl-1 mRNA level and Mcl-1 protein expressions 24 h and 48 h after the transfection ,and the silencing effects of the three pairs of specific shRNA fragments corresponding to different sites were analyzed.Results: Specific shRNA fragments at 24 h and 48 h could effectively reduce Mcl-1 mRNA and protein level ,with higher silencing effects than those of the normal group ,the lipofectamine group and the negative control group.There were statistically significant differences among them ( P<0.05 ).Among the three pairs of specific shRNA fragments corre-sponding to different sites ,Mcl-1 shRNA3 showed the most significant inhibiting effect on Mcl-1 mRNA and proteins.Conclusion:RNA interference can downregulate the level of Mcl-1 mRNA in murine macrophage cell line Raw 264.7 and greatly downregulate the expression of Mcl-1protein.Specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene have been screened out successfully.