OBJECTIVES: To investigate the role of the calcium/calmodulin (CaM)-mediated activation of calcineurin (CN)-nuclear factor of activated T cells (NFAT) signaling pathway in mediating the regulatory effect of hyperforin (HY) on stress-induced depression-like disorder (DP) in mice. METHODS: C57BL/6J mice were randomly divided into control group, DP model group, and hyperforin treatment group (n=15). Behavioral changes of the mice were assessed using open field test (OFT), sucrose preference test (SPT), tail suspension test (TST), light/dark box test (LDB), and novel object suppression test (NSFT). Immunohistochemistry was used to detect tyrosine hydroxylase (TH) expression in the CA1 region of the hippocampus, and serum serotonin (5-HT) and norepinephrine (NA) levels were detected with ELISA. Western blotting was used to analyze the expressions of TNF-α, IL-1β, IL-2, and CN-NFAT pathway proteins. In cultured BV-2 microglial cells with lipopolysaccharide (LPS) stimulation, the effects of hyperforin and CN inhibitor (CNIS) on expressions of ionized calcium-binding adapter molecule 1 (IBA-1), 5-HT, NA, inflammatory cytokines and CN-NFAT pathway proteins were examined using immunofluorescence assay, ELISA or Western blotting. RESULTS: Compared with the control mice, the mice in DP group showed significantly reduced activity in OFT, decreased sucrose consumption in SPT, reduced shuttle crossing in LDB, and lowered food intake in NSFT with significantly increased immobility in TST. The mice with DP showed significantly decreased TH-positive neurons, lowered 5-HT and NA levels, and increased expressions of TNF-α, IL-1β, IL-2 and CaM-CN-NFAT pathway proteins. In cultured BV-2 cells, LPS stimulation strongly increased cellular IBA-1 expression, decreased the levels of neurotransmitters (5-HT and NA), and increased the levels of inflammatory cytokines and CN-NFAT signaling, and these changes were effectively reversed by treatment with hyperforin or CNIS. CONCLUSIONS Hyperforin improves stress-induced depression-like behaviors in mice and activated BV-2 cells by targeting the CN-NFAT signaling pathway.
Animals ; Mice, Inbred C57BL ; Mice ; Microglia/drug effects* ; Depression/etiology* ; Perylene/pharmacology* ; Calcineurin/metabolism* ; NFATC Transcription Factors/metabolism* ; Calcium Signaling/drug effects* ; Stress, Psychological ; Phloroglucinol/pharmacology* ; Signal Transduction ; Male ; Behavior, Animal/drug effects* ; Terpenes

Objective To investigate the effect and mechanism of Congrong Shujing Granules on promoting microglial polarization and inhibiting neuroinflammation through the nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)/Caspase-1 signaling pathway.Methods Twenty Sprague-Dawley rats were assigned to the blank serum and Congrong Shujing Granules containing serum groups using random number table method,with 10 rats in each group.Rats in the Congrong Shujing Granules containing serum group received intragastric administration of Congrong Shujing Granules(2.57 g/kg)and the rats in the blank serum group received intragastric administration of physiological saline of equal volume.Blank serum and Congrong Shujing Granules containing serum were prepared separately.Mouse microglia cells BV-2 were cultured in vitro,and the optimal concentration of 1-methyl-4-phenylpyridine(MPP+)and optimal volume fraction of Congrong Shujing Granules containing serum were selected by the CCK-8 assay and immunofluorescence staining.And the NLRP3 inhibitor MCC950 was used as a postive control.Cells were divided into the blank serum group(10%blank serum),model group(10%blank serum+500 μmol/L MPP+),Congrong Shujing Granules containing serum group(10%Congrong Shujing Granules containing serum+500 μmol/L MPP+),and MCC950 group(10%blank serum+10 μmol/L MCC950+500 μmol/L MPP+),and intervened separately.After 14 h of intervention,morphological changes in BV-2 cells were observed.The contents of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6,and IL-4 were detected by an enzyme-linked immunosorbent assay.The mRNA expressions of differentiation cluster 86(CD86),inducible nitric oxide synthase(iNOS),CD206,and arginase 1(Arg1)were detected by real-time fluorescence PCR.The expressions of CD86,Arg1,Ionized calcium binding adaptor molecule 1(Iba1),and NLRP3 were detected by immunofluorescence staining.The expression of iNOS,Arg1,TNF-α,IL-6,IL-4,NLRP3,pro-cysteinyl aspartate specific proteinase 1(pro-Caspase-1),and Caspase-1 proteins was detected by Western blotting.Results Iba1 activation and expression increased under the MPP+(12 h,500 μmol/L)intervention(P<0.05),and cell viability was not affected.There was no statistically significant effect on cell viability after treatment with 10%Congrong Shujing Granules containing serum alone or in combination with MPP+(P>0.05).Compared to the blank serum group,BV-2 cells in the model group showed multiple branches and protruded in the shape of an amoeba.The contents of IL-1β,TNF-α,and IL-6 increased,while the contents of IL-4 decreased.The mRNA expressions of CD86 and iNOS increased,while mRNA expressions of CD206 and Arg1 decreased.The mean fluorescence intensity of CD86,Iba1,and NLRP3 increased,while the mean fluorescence intensity of Arg1 decreased.The protein expressions of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 increased,while the protein expressions of Arg1,IL-4 decreased,P<0.05.Compared to the model group,the Congrong Shujing Granules containing serum group and MCC950 group showed a decrease in the branch of cell protrusions,reduced cell activation,decreased levels of IL-1β,TNF-α,and IL-6,increased levels of IL-4,decreased expression of CD86 and iNOS mRNA,increased expression of CD206 mRNA,the decreased mean fluorescence intensity of CD86,Iba1,and NLRP3,the increased mean fluorescence intensity of Arg1,decreased expression of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 proteins,and increased expression of Arg1 and IL-4 proteins,P<0.05.Conclusion Congrong Shujing Granules containing serum may alleviate the MPP+-induced neuroinflammatory response by inhibiting the NLRP3/Caspase-1 signaling pathway to regulate M1/M2 phenotype polarization of microglia.

Objective To investigate the effect and mechanism of Congrong Shujing Granules on promoting microglial polarization and inhibiting neuroinflammation through the nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)/Caspase-1 signaling pathway.Methods Twenty Sprague-Dawley rats were assigned to the blank serum and Congrong Shujing Granules containing serum groups using random number table method,with 10 rats in each group.Rats in the Congrong Shujing Granules containing serum group received intragastric administration of Congrong Shujing Granules(2.57 g/kg)and the rats in the blank serum group received intragastric administration of physiological saline of equal volume.Blank serum and Congrong Shujing Granules containing serum were prepared separately.Mouse microglia cells BV-2 were cultured in vitro,and the optimal concentration of 1-methyl-4-phenylpyridine(MPP+)and optimal volume fraction of Congrong Shujing Granules containing serum were selected by the CCK-8 assay and immunofluorescence staining.And the NLRP3 inhibitor MCC950 was used as a postive control.Cells were divided into the blank serum group(10%blank serum),model group(10%blank serum+500 μmol/L MPP+),Congrong Shujing Granules containing serum group(10%Congrong Shujing Granules containing serum+500 μmol/L MPP+),and MCC950 group(10%blank serum+10 μmol/L MCC950+500 μmol/L MPP+),and intervened separately.After 14 h of intervention,morphological changes in BV-2 cells were observed.The contents of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6,and IL-4 were detected by an enzyme-linked immunosorbent assay.The mRNA expressions of differentiation cluster 86(CD86),inducible nitric oxide synthase(iNOS),CD206,and arginase 1(Arg1)were detected by real-time fluorescence PCR.The expressions of CD86,Arg1,Ionized calcium binding adaptor molecule 1(Iba1),and NLRP3 were detected by immunofluorescence staining.The expression of iNOS,Arg1,TNF-α,IL-6,IL-4,NLRP3,pro-cysteinyl aspartate specific proteinase 1(pro-Caspase-1),and Caspase-1 proteins was detected by Western blotting.Results Iba1 activation and expression increased under the MPP+(12 h,500 μmol/L)intervention(P<0.05),and cell viability was not affected.There was no statistically significant effect on cell viability after treatment with 10%Congrong Shujing Granules containing serum alone or in combination with MPP+(P>0.05).Compared to the blank serum group,BV-2 cells in the model group showed multiple branches and protruded in the shape of an amoeba.The contents of IL-1β,TNF-α,and IL-6 increased,while the contents of IL-4 decreased.The mRNA expressions of CD86 and iNOS increased,while mRNA expressions of CD206 and Arg1 decreased.The mean fluorescence intensity of CD86,Iba1,and NLRP3 increased,while the mean fluorescence intensity of Arg1 decreased.The protein expressions of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 increased,while the protein expressions of Arg1,IL-4 decreased,P<0.05.Compared to the model group,the Congrong Shujing Granules containing serum group and MCC950 group showed a decrease in the branch of cell protrusions,reduced cell activation,decreased levels of IL-1β,TNF-α,and IL-6,increased levels of IL-4,decreased expression of CD86 and iNOS mRNA,increased expression of CD206 mRNA,the decreased mean fluorescence intensity of CD86,Iba1,and NLRP3,the increased mean fluorescence intensity of Arg1,decreased expression of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 proteins,and increased expression of Arg1 and IL-4 proteins,P<0.05.Conclusion Congrong Shujing Granules containing serum may alleviate the MPP+-induced neuroinflammatory response by inhibiting the NLRP3/Caspase-1 signaling pathway to regulate M1/M2 phenotype polarization of microglia.

Objective To evaluate the diagnostic value of one-stop cardiac CT (stress dynamic CT-MPI combining with CTA) in CAD. Methods Thirty patients underwent CCTA combined with stress dynamic CT-MPI and stress SPECT-MPI including 16 patients underwent ICA were retrospectively enrolled. All procedures were performed within 2 weeks without related treatment. CT-MPI and SPECT-MPI were evaluated visually and MBF was quantified according to dynamic CT-MPI which was compared through paired t test. The diagnostic value of CT-MPI for myocardial ischemia was evaluated with SPECT-MPI as the reference standard. The consistency between the two techniques was determined by Kappa test. With SPECT combined with ICA as the reference standard, we evaluated the value of CT-MPI and its combination with CCTA in the diagnosis of CAD with hemodynamic stenosis. Results On stress CT-MPI, 19 of 30 patients and 52 segments were evaluated with myocardial perfusion defects. There was a significant difference in MBF values between normal[(117.61±29.82)ml·100 ml-1·min-1] and hypoperfused [(80.60±22.15)ml·100 ml - 1 · min - 1] segments (t=15.764,P<0.001). With SPECT as reference standard, the sensitivity, specificity were 94.12% (32/34), 95.80% (456/476) on the vessel basis. The Kappa value of the two examinations was 0.772(P<0.001). As for the 16 patients who also underwent ICA, 15 patients with 22 vessel-areas had hemodynamic stenosis on CT-MPI. Compared with SPECT combined with ICA, the sensitivity, specificity for detecting hemodynamic stenosis were 92.31% (12/13), 71.43% (25/35) for CT-MPI;100%(13/13), 51.43%(18/35) for CTA and 92.31%(12/13), 82.86%(29/35) for CT-MPI combined with CTA, respectively. Moderate correlation was observed between the combination of CT-MPI and CCTA and reference standard (r=0.690,P<0.001). Conclusion Stress dynamic CT-MPI can detect myocardial perfusion defects and hemodynamic changes and improves the diagnostic ability of functional stenosis of CAD combined with CTA, which can finally achieve the one-stop mode of cardiac CT in CAD.

Objective To evaluate the efficacy and safety of REXEEDTM series highflux dialyzer. Methods A randomized cross-over study of 3×3 Latin square was designed based on the surface area of dialyzer membrane (1.5 m2 and 2.1 m2). Seventy-two stable maintenance hemodialysis (MHD) patients from Shanghai Renji Hospital and Ruijin Hospital were enrolled in this study for 3 consecutive weeks. REXEEDTM-15AC, 15UC, 21AC, 21UC dialyzers were used as trial group and APS-15U,BIO-HX100 dialyzers were used as control group. The clearances of urea, creatinine, phosphorus and β2-microglobulin were calculated. Adverse event and adverse reactions were recorded. Results There were significantly higher urea and creatinine clearance in 1SAC and 15UC dialyzers as compared to APS-15U dialyzer [(222.07±18.74) mi/min, (220.23±26.26) ml/min vs (199.56±14.21) ml/min; (176.73±16.41) ml/min, (175.22±25.94) ml/min vs (165.42±14.68) ml/min, all P<0.05]. There were significantly higher urea, creatinine and β2-microglobulin clearance in 21AC and 21UC dialyzer as compared to BIO-HX100 dialyzer [(230.59±15.24) ml/min, (233.96±7.06) ml/min vs (203.43±36.66) ml/min; (183.50±25.90) ml/min, (181.05±23.94) ml/min vs (166.25±29.82) ml/min; (111.77±53.42) ml/min, (125.54±51.99) ml/min vs (42.39±4.81) ml/min; all P<0.05]. There was no significant difference of phosphorus clearance between REXEEDTM series dialyzers and control dialyzers. The efficiency of urea clearance and urea reduction ratio could achieve clinical targets in REXEEDTM series. Conclusion REXEEDTM series highflux dialyzers are effective and safe for clinical application.