The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.
Cells, Cultured ; Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Glucose/*pharmacology ; Immediate-Early Proteins/metabolism ; Immediate-Early Proteins/*physiology ; Mesangial Cells/cytology ; Mesangial Cells/*metabolism ; Protein-Serine-Threonine Kinases/metabolism ; Protein-Serine-Threonine Kinases/*physiology ; Signal Transduction/drug effects

Summary: The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.

ObjectiveTo establish a quantitative analysis multi-components by single marker method （QAMS） for five main components （aucubin， geniposidic acid， chlorogenic acid， asperuloside and rutin） in Eucommiae Folium， to verify its feasibility and applicability in the determination of Eucommiae Folium， so as to provide a scientific basis for the development of quality standard of this herb. MethodHigh performance liquid chromatography was performed on a Welch Boltmatetm™ C₁₈ column （4.6 mm×100 mm， 2.7 μm） with methanol （A）-0.2% phosphoric acid aqueous solution （B） as the mobile phase for gradient elution （0-8 min， 3%A； 8-10 min， 3%-11%A； 10-26 min， 11%A； 26-27 min， 11%-25%A； 27-60 min， 25%-32%A）， the column temperature was set at 30 ℃， the flow rate was 0.6 mL·min^-1， the detection wavelengths were at 210 nm and 254 nm. Chlorogenic acid was used as an internal reference to establish the relative correction factors （f） between it and the other four components， and the contents of the five components in 14 batches of Eucommiae Folium were determined by QAMS and external standard method （ESM）， respectively. ResultThe f values of chlorogenic acid to aucubin， geniposidic acid， asperuloside and rutin were 3.13， 1.45， 2.64 and 0.56， respectively. Repeatability was good under different experimental conditions， relative standard deviation （RSD） was <5.0%. The contents of aucubin， geniposidic acid， chlorogenic acid， asperuloside and rutin in 14 batches of Eucommiae Folium were 1.340-28.975， 0.252-36.086， 10.016-27.443， 1.396-8.646， 0.533-1.766 mg·g^-1， respectively. There were no significant difference between content results of QAMS and that of ESM （RSD<5.0%）. ConclusionQAMS established with chlorogenic acid as the internal reference can be used to determine the contents of five components in Eucommiae Folium， and this method is simple and accurate. After comprehensive evaluation， the quality standard of Eucommiae Folium in subsequent editions of Chinese Pharmacopoeia is suggested that three main active components， chlorogenic acid， aucubin and geniposidic acid， are selected as quality markers， and their content limits are recommended not less than 1.5%， 1.0% and 1.0%， respectively. This quality standard draft can avoid the potential quality risk due to poor specificity and low content limit of the index component （chlorogenic acid） in the previous editions of Chinese Pharmacopoeia.