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Aim To investigate the mechanism of eata- pol (CAT) inhibiting differentiation and glyeolysis of Thl7 eel Is through miR-143-3p.Method The peripheral hloorl CD4 ∗ T eells of HA patients were obtained to deteet the expression of miR-143-3p and the mRNA levels of key glycolytic enzymes, ineluding glucose transporter 1 ( Glutl ) , hexokinase 2 ( HK2 ) , pyruvate kinase 2 (PKM2) , laetate dehydrogenase A ( LDHA).The differentiation of Thl7 eells was induced in vitro, and the ShRNA/lentivirus was applied to achieve the overexpression or knockdown of miR- 143-3 p.Un-transfected eells were divided into control group and CAT group (20, 40, 80 mg • L 1 ) , and transfected eells were divided into four groups: negative control group, miR-143-3p inhibitor group, miR- 143-3p mimies group, miR-143-3p inhibitor + CAT group.The percentage of Thl7 eells was deteeted by flow cytometry, and the level of IL-17A was detected by EL1SA.Quantitative real-time PCR was used to detect the mRNA expression of miR-143-3p and key glycolytic enzymes, and the levels of pyruvate and lactate were also detected.Results The mRNA expression of miR-143-3p in RA peripheral blood CD4 ∗ T cells was negatively correlated with disease severity ( DAS28 ) , transcription factor ROR-yt, and the key glycolytic enzymes Glutl/HK2/LDHA.Compared with negative control group, the down-expression of miR-143-3p markedly elevated the mRNA expression of ROR-yt, Glutl, HK2, LDHA, and the levels of IL-17A, pyruvate, lactate.Catalpol groups significantly up-regula- ted the expression of miR-143-3p, decreased the mRNA expression of HK2/LDHA and the levels of pvru- vate/lactate, and inhibited Thl7 cells differentiation.Compared with miR - 1 4 3 - 3 p inhibitor group , catapol could significantly inhibit the abnormal up-regulated of HK2/LDHA mRNA relative expression, pyruvate/lactate levels and the abnormal differentiation of Thl7 eells.Conclusion MiR-143-3p inhibits the differentiation and glycolysis of Thl7 cells.Catalpol could sup-press the glycolysis and differentiation of Thl7 eells by regulating mill-143-3p.
RESUMO
Objective::To establish the HPLC fingerprint of carbonized ginger and to determine the contents of zingerone, 6-gingerol, 6-shogaol, 10-gingerol, 8-shogaol and 10-shogaol with quantitative analysis of multi-components by single marker (QAMS). Method::The fingerprint of carbonized ginger was established by HPLC. All samples were analyzed by Waters SymmetryShield™ RP18 column (4.6 mm×250 mm, 5 μm) with gradient elution by acetonitrile(A)-water(B) (0-30 min, 25%-70%A; 30-50 min, 70%-90%A; 50-60 min, 90%A), the flow rate was 1.0 mL·min-1, the detection wavelength was set at 240 nm and the column temperature was 30 ℃. Zingerone, 6-gingerol, 8-gingerol, 6-shogaol, 10-gingerol, 8-shogaol and 10-shogaol was chosen as marker ingredients to establish HPLC fingerprint of carbonized ginger decoction pieces. Taking 6-gingerol as internal reference standard, the contents of zingerone, 6-shogaol, 10-gingerol, 8-shogaol and 10-shogaol were determined at the detection wavelength of 220 nm and 280 nm according to the relative correction factor. Result::The HPLC fingerprint of carbonized ginger was obtained and 10 common peaks were designated, and 7 of them were identified as zingerone, 6-gingerol, 8-gingerol, 6-shogaol, 10-gingerol, 8-shogaol and 10-shogaol, respectively. And there were no significant differences between the quantitative results of external standard method and QAMS. It is suggested that the content limits of carbonized ginger should be not less than 0.020%of zingerone (C11H14O3), 0.050%of 6-gingerol (C17H26O4), 0.120%of 6-shogaol (C17H24O3), 0.080%of 10-gingerol (C21H34O4), 0.030%of 8-shogaol (C19H28O3) and 0.050%of 10-shogaol (C21H32O3) calculated with reference to the dried products, respectively. Conclusion::The developed method is accurate and feasible, which can provide a simple and effective method for the quality control of carbonized ginger.