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 explore the possibility of mutual regulation between TRiC/CCT complex(TCP-1 ring complex/chaperonin containing TCP-1)and mTOR complex 1(mechanistic target of rapamycin complex 1).Methods co-immunoprecipi-tation and Western blot were adopted to explore the interaction between TRiC/CCT complex and mTORC1.Rapamycin was used to inhibit the activity of mTORC 1.Soluble and insoluble cell protein samples were prepared,and the changes in protein ex-pression levels of TRiC/CCT complex-assisted folding were compared.The changes were observed via non-denaturing gel elec-trophoresis to explore the influences of mTORC1 on the integrity of TRiC/CCT complex.The CCT2 or CCT5 gene in HEK-293T cells was silenced by lentivirus-mediated shRNA to explore the influences of TRiC/CCT complex on the expression of key components of mTORC1 and the phosphorylation level of downstream substrates.Results Interaction between each subunit of TRiC/CCT complex and mTORC1 was proved by co-immunoprecipitation experiment.The activity of mTORC1 was inhibited by rapamycin.Expression levels of β-actin,β-Tubulin,and STAT3 insoluble proteins were not significantly increased,and the molecular size of the complex did not change significantly in the non-denaturing electrophoresis gel,indicating that mTORC1 had no regulative effect on formation of TRiC/CCT complex.CCT2 or CCT5 was silenced in HEK-293T.The protein expres-sion levels of mTOR,Raptor,and mLST8 were decreased,and the phosphorylation levels of p70S6K and Akt were decreased,re-vealing that the TRiC/CCT complex had a regulative effect on mTORC1 and downstream molecules.Conclusion The protein expression level of each component of mTORC1 was regulated by TRiC/CCT complex,thus affecting the phosphorylation level and activity status of the downstream substrates.

Objective To explore the possibility of mutual regulation between TRiC/CCT complex(TCP-1 ring complex/chaperonin containing TCP-1)and mTOR complex 1(mechanistic target of rapamycin complex 1).Methods co-immunoprecipi-tation and Western blot were adopted to explore the interaction between TRiC/CCT complex and mTORC1.Rapamycin was used to inhibit the activity of mTORC 1.Soluble and insoluble cell protein samples were prepared,and the changes in protein ex-pression levels of TRiC/CCT complex-assisted folding were compared.The changes were observed via non-denaturing gel elec-trophoresis to explore the influences of mTORC1 on the integrity of TRiC/CCT complex.The CCT2 or CCT5 gene in HEK-293T cells was silenced by lentivirus-mediated shRNA to explore the influences of TRiC/CCT complex on the expression of key components of mTORC1 and the phosphorylation level of downstream substrates.Results Interaction between each subunit of TRiC/CCT complex and mTORC1 was proved by co-immunoprecipitation experiment.The activity of mTORC1 was inhibited by rapamycin.Expression levels of β-actin,β-Tubulin,and STAT3 insoluble proteins were not significantly increased,and the molecular size of the complex did not change significantly in the non-denaturing electrophoresis gel,indicating that mTORC1 had no regulative effect on formation of TRiC/CCT complex.CCT2 or CCT5 was silenced in HEK-293T.The protein expres-sion levels of mTOR,Raptor,and mLST8 were decreased,and the phosphorylation levels of p70S6K and Akt were decreased,re-vealing that the TRiC/CCT complex had a regulative effect on mTORC1 and downstream molecules.Conclusion The protein expression level of each component of mTORC1 was regulated by TRiC/CCT complex,thus affecting the phosphorylation level and activity status of the downstream substrates.

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.

As the most aggressive breast cancer, triple-negative breast cancer (TNBC) is still incurable and very prone to metastasis. The transform growth factor β (TGF-β)-induced epithelial-mesenchymal transition (EMT) is crucially involved in the growth and metastasis of TNBC. This study reported that a natural compound isotoosendanin (ITSN) reduced TNBC metastasis by inhibiting TGF-β-induced EMT and the formation of invadopodia. ITSN can directly interact with TGF-β receptor type-1 (TGFβR1) and abrogated the kinase activity of TGFβR1, thereby blocking the TGF-β-initiated downstream signaling pathway. Moreover, the ITSN-provided inhibition on metastasis obviously disappeared in TGFβR1-overexpressed TNBC cells in vitro as well as in mice bearing TNBC cells overexpressed TGFβR1. Furthermore, Lys232 and Asp351 residues in the kinase domain of TGFβR1 were found to be crucial for the interaction of ITSN with TGFβR1. Additionally, ITSN also improved the inhibitory efficacy of programmed cell death 1 ligand 1 (PD-L1) antibody for TNBC in vivo via inhibiting the TGF-β-mediated EMT in the tumor microenvironment. Our findings not only highlight the key role of TGFβR1 in TNBC metastasis, but also provide a leading compound targeting TGFβR1 for the treatment of TNBC metastasis. Moreover, this study also points out a potential strategy for TNBC treatment by using the combined application of anti-PD-L1 with a TGFβR1 inhibitor.

Shengmai formula,composed of Ginseng Radix et Rhizoma,Ophiopogon Radix and Schisandrae Chinensis Fructus,is a classic and famous formula.It is a representative formula for"supplementing qi,nourishing yin,and generating fluid"in Traditional Chinese Medicine theory.To date,a wide range of Shengmai formulae have been developed according to different medical applications,but the quality evaluation standards are at a relatively low level,and most of them only specify the individual components of a single herb,making it difficult to ensure clinical efficacy and safety.At the same time,the physical and chemical identification methods of Shengmai formula have been constantly updated,allowing for greater progress in research on its main chemical components such as saponins,lignans and flavonoids.However,there is little systematic summarization of the chemical components and analytical methods.Based on the existing references,we systematically summarized ginsenosides,ophiopogonins,schisandra lignans,homoisoflavonoids and some other compounds in this paper,as well as the quality standards of Shengmai formulae and their analytical methods in order to aid clinical research and formulation manufacture.

Plantamajoside is one of the main bioactive compounds in Plantaginis Herba A TLC method was developed identification of plantamajoside in 11 Plantaginis Herba samples using silica gel G as coating substance and a mixture of ethyl acetiate methanol-formic acid-water (18: 3 : 1.5 : 1) as a developing solvent, the established TLC condition displayed a very well separation on the chromatogram of tested Plantaginis Herba samples and the marker compound plantamajoside showed as a distinct light-blue fluorescence spot observed under UV 365 nm. Using the HPLC method, plantamajoside was separated at 30 degrees C on a Promocil C18, (4.6 mm x 250 mm, 5 microm) column with acetonitrile-0.1% formic acid (17:83) as the mobile phase. The detection wavelength was set at 330 nm and the flow rate was 1 mL x min(-1). The calibration curve of plantamajoside displayed ideal linearity over the range of 0.0499-11.9664 microg (r = 0.9999), and the average recovery of plantamajoside was 100.6% with a RSD of 2.7%. The contents of plantamajoside were in the range of 0.067%-1.80% in Plantaginis Herba The established TLC identification and HPLC were sensitive, reliable and repeatable, which can be applied for the quality evaluation and standard criteria of Plantaginis Herba.
Asteraceae ; chemistry ; Catechols ; analysis ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Glucosides ; analysis ; Reproducibility of Results