Objective:To investigate protective effect and mechanism of Tim-3 on sepsis-induced acute lung injury(ALI)by pro-moting mitophagy of alveolar macrophages and inhibiting activation of NLRP3 inflammasome.Methods:LPS-stimulated mouse alveo-lar macrophage(MH-S)model and sepsis-induced ALI mouse model were constructed.Tim-3 siRNA interference technique was used to knock down Tim-3 expression in MH-S cells,and anti-Tim-3 antibody mice were injected intraperitoneally to block Tim-3 function.Western blot was used to detect protein expressions of NLRP3,ASC,cleaved-caspase-1 and mitophagy-related proteins(LC3B,P62,PINK1 and Parkin)in MH-S cells and lung tissue of mice with sepsis-induced ALI.Laser confocal fluorescence staining was used to measure ROS level and mitochondrial membrane potential of MH-S cells.Pathological examination of lung tissue was performed in mice with sepsis-induced ALI in each group,and degree of lung tissue injury was evaluated by Smith scoring system.Bronchoalveolar lavage fluid(BALF)and lung tissue were collected from mice with ALI induced by sepsis in each group.BCA protein quantification method was used to determine protein concentration in BALF.MPO activity in lung tissue was detected by colorimetry.MDA content in lung tissue was detected by TBA method.LC3B protein expression in lung tissue was detected by immunohistochemistry.Results:In mouse alveolar macrophages,Tim-3 knockdown could promote expressions of NLRP3,ASC,cleaved-caspase-1 and P62 proteins,increase ROS release,inhibit PINK1/Parkin pathway activation and LC3B protein expression,and reduce mitochondrial membrane potential.In mice with sepsis-induced ALI,Tim-3 functional blockade could promote expressions of NLRP3,ASC,cleaved-caspase-1 and P62 proteins in lung tissue,aggravate lung pathological injury and pulmonary edema,increase MPO activity and MDA content in lung tissue,and reduce positive rate of LC3B protein.Conclusion:Tim-3 plays a protective role in sepsis-induced ALI by promoting mitophagy in alveolar macrophages and inhibiting NLRP3 inflammasome activation via PINK1/Parkin.

OBJECTIVE To evaluate the immunoprotection effect of a novel inactivated whole cell vaccine against Acinetobacter baumannii based on ultrasonic microbubble physical damage technique(IWC)and explore its poten-tial of clinical transformation.METHODS Totally 48 C57BL/6 mice were randomly assigned to divide into three groups and receive the nasal inoculation of corresponding preparations,the IWC group and the paraformalde-hyde inactivated vaccine group were inoculated with 20 μl of 1× 107 CFU vaccine,the control group was treated with 20 μl phosphate buffered salt solution.The infection models were established 7 days after intraperitoneal in-jection of a lethal dose of A.baumannii.The 7-day mortality rates of the mice were statistically analyzed after tox-in attack.The counts of colonized bacterial colonies on lung and spleen tissues were determined by plate count method after toxin attack for 24 hours.The levels of inflammatory factors interleukin(IL)-6,tumor necro-sis factor α(TNF-α)and IL-1β in the lung tissues were detected by enzyme-linked immunosorbent assay(ELISA),and the pathological damage was observed.RESULTS The survival rate of the IWC group was higher than that of the control group,and the counts of colonized bacterial colonies on lung and spleen tissues were less in the IWC group than those in the control group(P＜0.05).As compared the paraformaldehyde inactivated vaccine group,the survival rate of the IWC group increased by 10.00％,and the counts of colonized bacterial colonies on the lung tissues were slightly less in the IWC group than those in the paraformaldehyde inactivated vaccine group(P＜0.05),and the counts of colonized bacterial colonies on spleens were basically the same.The levels of lung tis-sue inflammatory factors of the IWC group were lower than those of the other two groups(P＜0.05).The patho-logical damage was alleviated,and the IWC group was superior to the control group in the integrity of alveolar structure.CONCLUSIONS IWC can maintain the immunogenicity of pathogens through physical damage technique,effectively activate the immune response of the hose,and reduce the bacterial load and inflammatory injury,show-ing better immunoprotection effect than the traditional chemical inactivation method.The study has provided ex-perimental bases for development of novel,specific,safe and highly efficient vaccine as well as new ideas and strategies for clinical prevention and treatment of A.baumannii infection.

Objective:To investigate protective effect and mechanism of Tim-3 on sepsis-induced acute lung injury(ALI)by pro-moting mitophagy of alveolar macrophages and inhibiting activation of NLRP3 inflammasome.Methods:LPS-stimulated mouse alveo-lar macrophage(MH-S)model and sepsis-induced ALI mouse model were constructed.Tim-3 siRNA interference technique was used to knock down Tim-3 expression in MH-S cells,and anti-Tim-3 antibody mice were injected intraperitoneally to block Tim-3 function.Western blot was used to detect protein expressions of NLRP3,ASC,cleaved-caspase-1 and mitophagy-related proteins(LC3B,P62,PINK1 and Parkin)in MH-S cells and lung tissue of mice with sepsis-induced ALI.Laser confocal fluorescence staining was used to measure ROS level and mitochondrial membrane potential of MH-S cells.Pathological examination of lung tissue was performed in mice with sepsis-induced ALI in each group,and degree of lung tissue injury was evaluated by Smith scoring system.Bronchoalveolar lavage fluid(BALF)and lung tissue were collected from mice with ALI induced by sepsis in each group.BCA protein quantification method was used to determine protein concentration in BALF.MPO activity in lung tissue was detected by colorimetry.MDA content in lung tissue was detected by TBA method.LC3B protein expression in lung tissue was detected by immunohistochemistry.Results:In mouse alveolar macrophages,Tim-3 knockdown could promote expressions of NLRP3,ASC,cleaved-caspase-1 and P62 proteins,increase ROS release,inhibit PINK1/Parkin pathway activation and LC3B protein expression,and reduce mitochondrial membrane potential.In mice with sepsis-induced ALI,Tim-3 functional blockade could promote expressions of NLRP3,ASC,cleaved-caspase-1 and P62 proteins in lung tissue,aggravate lung pathological injury and pulmonary edema,increase MPO activity and MDA content in lung tissue,and reduce positive rate of LC3B protein.Conclusion:Tim-3 plays a protective role in sepsis-induced ALI by promoting mitophagy in alveolar macrophages and inhibiting NLRP3 inflammasome activation via PINK1/Parkin.

OBJECTIVE To evaluate the immunoprotection effect of a novel inactivated whole cell vaccine against Acinetobacter baumannii based on ultrasonic microbubble physical damage technique(IWC)and explore its poten-tial of clinical transformation.METHODS Totally 48 C57BL/6 mice were randomly assigned to divide into three groups and receive the nasal inoculation of corresponding preparations,the IWC group and the paraformalde-hyde inactivated vaccine group were inoculated with 20 μl of 1× 107 CFU vaccine,the control group was treated with 20 μl phosphate buffered salt solution.The infection models were established 7 days after intraperitoneal in-jection of a lethal dose of A.baumannii.The 7-day mortality rates of the mice were statistically analyzed after tox-in attack.The counts of colonized bacterial colonies on lung and spleen tissues were determined by plate count method after toxin attack for 24 hours.The levels of inflammatory factors interleukin(IL)-6,tumor necro-sis factor α(TNF-α)and IL-1β in the lung tissues were detected by enzyme-linked immunosorbent assay(ELISA),and the pathological damage was observed.RESULTS The survival rate of the IWC group was higher than that of the control group,and the counts of colonized bacterial colonies on lung and spleen tissues were less in the IWC group than those in the control group(P＜0.05).As compared the paraformaldehyde inactivated vaccine group,the survival rate of the IWC group increased by 10.00％,and the counts of colonized bacterial colonies on the lung tissues were slightly less in the IWC group than those in the paraformaldehyde inactivated vaccine group(P＜0.05),and the counts of colonized bacterial colonies on spleens were basically the same.The levels of lung tis-sue inflammatory factors of the IWC group were lower than those of the other two groups(P＜0.05).The patho-logical damage was alleviated,and the IWC group was superior to the control group in the integrity of alveolar structure.CONCLUSIONS IWC can maintain the immunogenicity of pathogens through physical damage technique,effectively activate the immune response of the hose,and reduce the bacterial load and inflammatory injury,show-ing better immunoprotection effect than the traditional chemical inactivation method.The study has provided ex-perimental bases for development of novel,specific,safe and highly efficient vaccine as well as new ideas and strategies for clinical prevention and treatment of A.baumannii infection.

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: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.

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.