Objective To observe the effect of Kaixin Powder on neuroinflammation in APP/PS1 mice by regulating WDFY1/TLR4/NF-κB signaling pathway;To explore its mechanism of intervening in Alzheimer disease(AD).Methods APP/PS1 transgenic mice were randomly divided into model group,donepezil hydrochloride group(0.66 mg/kg),and Kaixin Powder low-,medium-and high-dosage groups(1.625,3.25,6.5 g/kg),C57BL/6J mice were set as blank control group,with 8 mice in each group,and corresponding drug intervention was given to medicaction group for 24 weeks.Morris water maze,Y maze and novel object recognition experiments were conducted to assess the cognitive function and learning and memory abilities of mice,immunohistochemical staining was used to detect the deposition of β-amyloid protein(Aβ)in hippocampus,the morphology and Nissl bodies of hippocampal CA1 neurons were observed using HE staining and Nissl staining,ELISA was used to detect the serum contents of interleukin(IL)-6,IL-17,IL-1β and tumor necrosis factor-α(TNF-α),Western blot was used to detect the protein expression of calcium-binding adapter molecule 1(Iba1),glial fibrillary acidic protein(GFAP),WDFY1,Toll like receptor 4(TLR4),Toll like receptor associated molecule(TRAM),TIR domain adapter protein(TRIF),NF-κB p65 and p-NF-κB p65 in hippocampal tissue,RT-qPCR was used to detect the mRNA expression of WDFY1,TLR4,TRAM,TRIF and NF-κB p65 in hippocampal tissue.Results Compared with the blank control group,the model group had significantly prolonged escape latency,reduced platform crossings,decreased autonomous reaction alternation rate and relative recognition index(P<0.05,P<0.01),with increased deposition of Aβ in hippocampal tissue(P<0.01),damaged morphological structure of neurons,reduced number of neurons and Nissl bodies,the serum contents of IL-6,IL-17,IL-1β and TNF-α significantly increased,the expression of Iba1,GFAP,WDFY1,TLR4,TRAM,TRIF,p-NF-κB p65 protein and WDFY1,TLR4,TRAM,TRIF mRNA in hippocampal tissue significantly increased(P<0.01).Compared with the model group,Kaixin Powder groups and donepezil hydrochloride group had significantly shortened escape latency and increased platform crossings,autonomous reaction alternation rate and relative recognition index(P<0.05,P<0.01),hippocampal Aβ deposition reduced in Kaixin Powder medium-,high-dosage groups and donepezil hydrochloride group,the morphological structure of neurons recovered,the number of neurons and Nissl bodies increased,the serum contents of IL-6,IL-17,IL-1β and TNF-α significantly decreased(P<0.05,P<0.01),and the protein expression of Iba1,GFAP,WDFY1,TLR4,TRAM,TRIF,p-NF-κB p65 and the mRNA expressions of WDFY1,TLR4,TRAM and TRIF in hippocampal tissue significantly decreased(P<0.05,P<0.01).Conclusion Kaixin Powder can improve cognitive function and learning and memory abilities in AD model mice,alleviate hippocampal neuron damage and Aβ deposition,inhibit the activation of microglia and astrocytes,and thereby reduce serum inflammatory cytokine release.Its mechanism may be related to regulating the WDFY1/TLR4/NF-κB signaling pathway to inhibit neuroinflammation.

ObjectiveTo investigate the mechanism of action of Kaixinsan in the treatment of Alzheimer's disease （AD） based on network pharmacology， molecular docking， and animal experimental validation. MethodsThe Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） and the Encyclopedia of Traditional Chinese Medicine（ETCM） databases were used to obtain the active ingredients and targets of Kaixinsan. GeneCards， Online Mendelian Inheritance in Man（OMIM）， TTD， PharmGKB， and DrugBank databases were used to obtain the relevant targets of AD. The intersection （common targets） of the active ingredient targets of Kaixinsan and the relevant targets of AD was taken， and the network interaction analysis of the common targets was carried out in the STRING database to construct a protein-protein interaction（PPI） network. The CytoNCA plugin within Cytoscape was used to screen out the core targets， and the Metascape platform was used to perform gene ontology（GO） functional enrichment analysis and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analysis. The “drug-active ingredient-target” interaction network was constructed with the help of Cytoscape 3.8.2， and AutoDock Vina was used for molecular docking. Scopolamine （SCOP） was utilized for modeling and injected intraperitoneally once daily. Thirty-two male C57/BL6 mice were randomly divided into blank control （CON） group （0.9% NaCl， n=8）， model （SCOP） group （3 mg·kg^-1·d^-1， n=8）， positive control group （3 mg·kg^-1·d^-1 of SCOP+3 mg·kg^-1·d^-1 of Donepezil， n=8）， and Kaixinsan group （3 mg·kg^-1·d^-1 of SCOP+6.5 g·kg^-1·d^-1 of Kaixinsan， n=8）. Mice in each group were administered with 0.9% NaCl， Kaixinsan， or Donepezil by gavage twice a day for 14 days. Morris water maze experiment was used to observe the learning memory ability of mice. Hematoxylin-eosin （HE） staining method was used to observe the pathological changes in the CA1 area of the mouse hippocampus. Enzyme linked immunosorbent assay（ELISA） was used to determine the serum acetylcholine （ACh） and acetylcholinesterase （AChE） contents of mice. Western blot method was used to detect the protein expression levels of signal transducer and activator of transcription 3（STAT3） and nuclear transcription factor（NF）-κB p65 in the hippocampus of mice. ResultsA total of 73 active ingredients of Kaixinsan were obtained， and 578 potential targets （common targets） of Kaixinsan for the treatment of AD were screened out. Key active ingredients included kaempferol， gijugliflozin， etc.. Potential core targets were STAT3， NF-κB p65， et al. GO functional enrichment analysis obtained 3 124 biological functions， 254 cellular building blocks， and 461 molecular functions. KEGG pathway enrichment obtained 248 pathways， mainly involving cancer-related pathways， TRP pathway， cyclic adenosine monophosphate（cAMP） pathway， and NF-κB pathway. Molecular docking showed that the binding of the key active ingredients to the target targets was more stable. Morris water maze experiment indicated that Kaixinsan could improve the learning memory ability of SCOP-induced mice. HE staining and ELISA results showed that Kaixinsan had an ameliorating effect on central nerve injury in mice. Western blot test indicated that Kaixinsan had a down-regulating effect on the levels of NF-κB p65 phosphorylation and STAT3 phosphorylation in the hippocampal tissue of mice in the SCOP model. ConclusionKaixinsan can improve the cognitive impairment function in SCOP model mice and may reduce hippocampal neuronal damage and thus play a therapeutic role in the treatment of AD by regulating NF-κB p65， STAT3， and other targets involved in the NF-κB signaling pathway.

Objective To observe the effect of Kaixin Powder on neuroinflammation in APP/PS1 mice by regulating WDFY1/TLR4/NF-κB signaling pathway;To explore its mechanism of intervening in Alzheimer disease(AD).Methods APP/PS1 transgenic mice were randomly divided into model group,donepezil hydrochloride group(0.66 mg/kg),and Kaixin Powder low-,medium-and high-dosage groups(1.625,3.25,6.5 g/kg),C57BL/6J mice were set as blank control group,with 8 mice in each group,and corresponding drug intervention was given to medicaction group for 24 weeks.Morris water maze,Y maze and novel object recognition experiments were conducted to assess the cognitive function and learning and memory abilities of mice,immunohistochemical staining was used to detect the deposition of β-amyloid protein(Aβ)in hippocampus,the morphology and Nissl bodies of hippocampal CA1 neurons were observed using HE staining and Nissl staining,ELISA was used to detect the serum contents of interleukin(IL)-6,IL-17,IL-1β and tumor necrosis factor-α(TNF-α),Western blot was used to detect the protein expression of calcium-binding adapter molecule 1(Iba1),glial fibrillary acidic protein(GFAP),WDFY1,Toll like receptor 4(TLR4),Toll like receptor associated molecule(TRAM),TIR domain adapter protein(TRIF),NF-κB p65 and p-NF-κB p65 in hippocampal tissue,RT-qPCR was used to detect the mRNA expression of WDFY1,TLR4,TRAM,TRIF and NF-κB p65 in hippocampal tissue.Results Compared with the blank control group,the model group had significantly prolonged escape latency,reduced platform crossings,decreased autonomous reaction alternation rate and relative recognition index(P<0.05,P<0.01),with increased deposition of Aβ in hippocampal tissue(P<0.01),damaged morphological structure of neurons,reduced number of neurons and Nissl bodies,the serum contents of IL-6,IL-17,IL-1β and TNF-α significantly increased,the expression of Iba1,GFAP,WDFY1,TLR4,TRAM,TRIF,p-NF-κB p65 protein and WDFY1,TLR4,TRAM,TRIF mRNA in hippocampal tissue significantly increased(P<0.01).Compared with the model group,Kaixin Powder groups and donepezil hydrochloride group had significantly shortened escape latency and increased platform crossings,autonomous reaction alternation rate and relative recognition index(P<0.05,P<0.01),hippocampal Aβ deposition reduced in Kaixin Powder medium-,high-dosage groups and donepezil hydrochloride group,the morphological structure of neurons recovered,the number of neurons and Nissl bodies increased,the serum contents of IL-6,IL-17,IL-1β and TNF-α significantly decreased(P<0.05,P<0.01),and the protein expression of Iba1,GFAP,WDFY1,TLR4,TRAM,TRIF,p-NF-κB p65 and the mRNA expressions of WDFY1,TLR4,TRAM and TRIF in hippocampal tissue significantly decreased(P<0.05,P<0.01).Conclusion Kaixin Powder can improve cognitive function and learning and memory abilities in AD model mice,alleviate hippocampal neuron damage and Aβ deposition,inhibit the activation of microglia and astrocytes,and thereby reduce serum inflammatory cytokine release.Its mechanism may be related to regulating the WDFY1/TLR4/NF-κB signaling pathway to inhibit neuroinflammation.

Objective: To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with cation exchange-based solid phase extraction (SPE) for determination of tetrodotoxin (TTX) in salted pufferfish. Methods: Evenly crushed salted pufferfish samples were subjected to ultrasound-assisted extraction with 0.5% acetic acid/50% methanol/water. The extract was cleaned with cation exchange-based SPE cartridge and eluted with 0.3% hydrochloric acid and 50% acetonitrile/water. The eluent was neutralized with ammonia and separated with a Waters XBridgeTM BEH Amide column (150 mm×3.0 mm, 1.7 μm), and determined using LC-MS/MS in a multiple reaction monitoring mode. Results: The matrix effects of TTX were 85.7%-92.4%, and the matrix suppression effect was under effective control following clean-up procedures using the optimized SPE method. The TTX showed a good linear relationship at the range of 2.0 to 4 000 μg/kg, with a correlation coefficient (r2) of 0.999 2. The limits of detection and quantitation for TTX in sample matrix were 1.0 μg/kg and 2.0 μg/kg, respectively. The mean spiked-recovery rates were 81.2% to 96.5% at spiked amounts of 2.0, 200 μg/kg and 2 200 μg/kg, with relative standard deviations (RSDs) of 4.3% to 7.5%. The intraday accuracy and precision of TTX were 84.4% to 95.6% and 4.9% to 5.8% in quality control samples, and the interday accuracy and precision of TTX were 86.1% to 94.9% and 5.5% to 8.5% in quality control samples. The detection of TTX was 60.5% in 38 market-sold salted pufferfish products using the established LC-MS/MS method. Conclusion The established LC-MS/MS method is effective for accurate quantitative determination of TTX in salted pufferfish.

BACKGROUND:Side population(SP)cells,with varied contents,are widely distributed in adult tissues,embryos,and certain tumor cells.Haematological tumor is one of the main pathological conditions,which endangers human life.Thus,it is important to recognize SP cells in haematological tumor cell lines.OBJECTIVE:To identify whether hematologic tumor cell lines contain SP cells,and to explore a stable detection and separation methods.METHODS:Cells with the characteristics of stem cells being capable of fluorescent dye Hoechst33342 were isolated by flow cytometry;whether there were SP cells in logarithmic growth period of NB4,Raji,K562/ADM and K562 or not were detected.After sorting K562 subpopulations,the purity of sorted cells was detected.RESULTS AND CONCLUSION:After Hoechst33342 staining,flow cytometry results showed that the SP cells appeared in the haematological tumor NB4,Raji,K562/ADM and K562 cell lines,which accounted for 0.8%,2.7%,1.3% and 2.7%,respectively.These cells could be blocked by Verapamil.The purity was greater after a second detection of SP and Non SP cells in K562 cells.

Objective To set up real-time quantitative RT-PCR technique and measure leukemia fusion gene transcripts in patients with AML of FAB-M_2 subtype,and also to investigate the positive rate in patients and the relationship between the AMLI/ETO mRNA levels and the response rate after chemical therapy.Methods The plasmid containing the AMLI/ETO fusion gene sequences were constructed from myeloid cell lines Kasumi-1 (expressing AML1/ETO)to establish the standard curves,A TaqMan based realtime quantitative RT-PCR was performed to measure aberrant fusion gene transcripts in 45 samples of peripheral blood(PB) or bone marrow (BM) from 25 newly diagnosed patients with AML-M_2.All these 25 patients were diagnosed by the FCM(flow cytometry)and bone marrow molecular cytogenetics,and received the induce remission therapy with MA (Mitoxantrone+Ara-C).Results As a result,the AML1/ETO fusion gene transcripts were detected in 7(28%)out of 25 AML-M_2 patients (the ratios of AML1/ETO/ABL vary from 0.01 to 19.2),in which 5 patients were found t(8;21)(q22;q22).The transcript level of AML1/ETO fusion gene varied from the clinical situation of patients.These 7 patients with AML1/ETO fusion gene got complete remission(CR) after the first MA therapy,and the fusion gene reduced by 3 log in AML1/ETO/ABL.Only 11 patients got CR in 18 patients without AML1/ETO fusion gene.By following up these 7 patients with AML1/ ETO fusion gene kept persistent CR for 6 months.Conclusion It was concluded that real-time quantitative PCR is a reliable,innovative and promising technology with high sensitivity and speciality.It has potential clinical value for diagnosis,tumor typing,treatment selection,measuring the tumor load,monitoring fusion gene expression level and evaluating therapeutic strategy.It is worthy to apply in the clinical practice.

Objective To investigate the effect of fetoscopic photocoagulation of communicating placental vessels in twin-twin transfusion syndrome(TTTS)(selective or non-selective) on the perinatal outcomes.Methods Six cases of TTTS admitted in our department from Dec.2006 to Jun.2008 underwent fetoscopic photocoagulation of communicating vessels.Under direct real-time sonographic guidance,a 3-mm-diameter fetoscope was percutaneously inserted through the maternal abdominal wall into the amniotic cavity of the recipient twin.A combination of ultrasonographic and fetoscopic vision was used to identify the crossing vessels which were systematically coagulated using Nd:YAG laser fiber or bipolar electrocoagulation.Results All the 6 mothers tolerated the procedure without major complications.Two fetal survival rate was 33.33%.Conclusion Fetoscopic photocoagulation of communicating placental vessels in TTTS can effectively improve perinatal outcomes.