ABSTRACT
OBJECTIVE@#To explore the effects of taurolithocholic acid (tLCA) and chenodeoxycholic acid (CDCA) on the expression of aorexigenic neuropeptide in mouse hypothalamus GT1-7 cells.@*METHODS@#Mouse hypothalamic GT1-7 cells were treated with culture medium containing 10% FBS (control group, =3) or with 10 nmol/L, 100 nmol/L, 1 μmol/L and 10 μmol/L tLCA (tLCA group, =3) or CDCA (CDCA group, =3) for 12, 24 or 48 h. Real-time PCR was performed to determine the expression levels of proopiomelanocortin (POMC) mRNA in the cells, and the production levels of α-melanocyte-stimulating hormone (α-MSH) were assessed using an ELISA kit. Signal transduction and activator of transcription 3 phosphorylation (p-STAT3), threonine kinase phosphorylation (p-AKT), suppressor of cytokine signaling 3 (SOCS3), G protein-coupled bile acid receptor-1 (TGR5) and farnesoid X receptor (FXR) protein were detected by Western blotting.@*RESULTS@#Western blotting results showed that mouse hypothalamic GT1-7 cells expressed two bile acid receptors, TGR5 and FXR, whose expressions were regulated by bile acids. Real-time PCR showed that the expression of POMC mRNA was significantly increased in the cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. POMC-derived anorexigenic peptide α-MSH increased significantly in GT1-7 cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. Treatment of the cells with tLCA or CDCA significantly increased the expressions of intracellular signaling proteins including p-STAT3, p-AKT and SOCS3.@*CONCLUSIONS@#Mouse hypothalamic GT1-7 cells express bile acid receptors TGR5 and FXR. Bile acids tLCA or CDCA can promote the expression of POMC mRNA and increase the production of the anorexigenic peptide α-MSH. The intracellular signaling proteins p-AKT, p-STAT3 and SOCS3 are likely involved in bile acid-induced anorexigenic peptide production.
Subject(s)
Animals , Mice , Bile Acids and Salts , Chenodeoxycholic Acid , Hypothalamus , Neuropeptides , Phosphorylation , STAT3 Transcription Factor , Signal Transduction , Suppressor of Cytokine Signaling 3 ProteinABSTRACT
OBJECTIVE@#To explore the effects of taurolithocholic acid (tLCA) and chenodeoxycholic acid (CDCA) on the expression of aorexigenic neuropeptide in mouse hypothalamus GT1-7 cells.@*METHODS@#Mouse hypothalamic GT1-7 cells were treated with culture medium containing 10% FBS (control group, =3) or with 10 nmol/L, 100 nmol/L, 1 μmol/L and 10 μmol/L tLCA (tLCA group, =3) or CDCA (CDCA group, =3) for 12, 24 or 48 h. Real-time PCR was performed to determine the expression levels of proopiomelanocortin (POMC) mRNA in the cells, and the production levels of α-melanocyte-stimulating hormone (α-MSH) were assessed using an ELISA kit. Signal transduction and activator of transcription 3 phosphorylation (p-STAT3), threonine kinase phosphorylation (p-AKT), suppressor of cytokine signaling 3 (SOCS3), G protein-coupled bile acid receptor-1 (TGR5) and farnesoid X receptor (FXR) protein were detected by Western blotting.@*RESULTS@#Western blotting results showed that mouse hypothalamic GT1-7 cells expressed two bile acid receptors, TGR5 and FXR, whose expressions were regulated by bile acids. Real-time PCR showed that the expression of POMC mRNA was significantly increased in the cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. POMC-derived anorexigenic peptide α-MSH increased significantly in GT1-7 cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. Treatment of the cells with tLCA or CDCA significantly increased the expressions of intracellular signaling proteins including p-STAT3, p-AKT and SOCS3.@*CONCLUSIONS@#Mouse hypothalamic GT1-7 cells express bile acid receptors TGR5 and FXR. Bile acids tLCA or CDCA can promote the expression of POMC mRNA and increase the production of the anorexigenic peptide α-MSH. The intracellular signaling proteins p-AKT, p-STAT3 and SOCS3 are likely involved in bile acid-induced anorexigenic peptide production.
Subject(s)
Animals , Mice , Cell Line , Chenodeoxycholic Acid , Pharmacology , Gene Expression Regulation , Hypothalamus , Cell Biology , Neuropeptides , Genetics , Metabolism , Pro-Opiomelanocortin , Genetics , RNA, Messenger , Genetics , STAT3 Transcription Factor , Metabolism , Signal Transduction , Suppressor of Cytokine Signaling 3 Protein , Metabolism , Taurolithocholic Acid , Pharmacology , alpha-MSH , GeneticsABSTRACT
Millettia pinnata L. is a leguminous tree with great potential in biodiesel applications and also a typical semi-mangrove. In this review, we presented several aspects about the recent research progress in molecular biology of M. pinnata. We descrived several types of molecular markers used to assess the genetic diversity and phylogeny of this species, genome and transcriptome analyses based on high-throughput sequencing platform accomplished for this species, and several gene and genomic sequences of this species isolated for further research. Finally, based on the current research progress, we proposed some orientations for future molecular biology research on M. pinnata.
Subject(s)
Base Sequence , Gene Expression Profiling , Genetic Variation , Genome, Plant , Genomics , Millettia , Genetics , Phylogeny , Trees , GeneticsABSTRACT
Objective To construct and identify recombinant expression plasmid of small interfering RNA (siRNA)targeting hepatitis B virus X protein(HBx), and observe its effect on mitoehondrial function in healthy liver cell line steadily expressed HBx gene (HL-7702/HBx). Methods Two siRNA sequences containing short hairpin structure, which target on the total length HBx gene, were synthesized and cloned into the vector psiRNA-Hh1GFPzeo to eonstruct recombinant expression plasmids pX1 and pX2. Non-specific recombinant pScr plasmid served as control. After siRNA transfected into HL-7702/HBx cells line by liposome, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to identify the suppressive effect on HBx expression. Levels of intraeellular reactive oxygen species (ROS) and mitochondrial membrane potential (△ m) were determined by flow cytometry. The experimental results were compared by analysis of variance. Results Successful constructions of pX1 and pX2 were confirmed by restriction enzyme digestion and sequencing. The expressions of HBx mRNA and protein after 48 h of transfection into HL-7702/HBx cells in control group were 0.65± 0.12 and 0.62± 0.09, respectively, which were both higher than those (0.33±0.10 and 0.19±0.08, respectively) in group pX1 (t=4.73, P<0.05; t=7.53, P<0.05) and those (0.48±0.10 and 0.37±0.11, respectively) in group pX2 (t=2.39, P<0.05;t=4.43,P<0.05). But the inhibition of group pX1 was stronger than that of pX2 (t=2.28,P<0.05). Levels of ROS and △ m after RNA interference were 5.00±0.38 and 33.86±0.50, respectively, while those in control group were 72. 10±0. 55 and 3. 57±0.26, respectively (ROS: t=276.22, P<0.05; △ m: t=107.15, P<0.05). Conclusions siRNA targeting HBx can efficiently and specifically suppress the HBx expression in HL-7702/HBx cells, and decrease the level of ROS and increase the level of △ m, thus relieve cellular oxidative stress.