Design, synthesis and anti-NASH effect evaluation of novel GFT505 derivatives in vitro and in vivo.

Non-alcoholic fatty liver disease (NAFLD) is emerging as the largest burden of chronic liver disease worldwide. Nonalcoholic steatohepatitis (NASH) is a progressive form of NAFLD that can progress to cirrhosis and hepatocellular carcinoma. Unfortunately, current treatment options for NASH are very limited. Among the multiple pathways of NASH, peroxisome proliferators-activated receptors (PPARS) are recognized as an important and effective target. GFT 505 is a dual excitement agent for the treatment of PPAR-α/δ for the treatment of NASH. However, its activity and toxicity need to be further improved. Therefore, here we would like to report the design, synthesis and biological evaluation of 11 GFT 505 derivatives. The initial cytotoxicity through proliferation activity of HepG2 cells and in vitro anti-NASH activity evaluation demonstrated that under the same concentration, the compound 3d possess significantly lower cytotoxicity and better anti-NASH activity than that of GFT 505. Moreover, Molecular docking also shows that 3d and PPAR-α/δ can form a stable hydrogen bond and have the lowest binding energy. Therefore this novel molecule 3d was selected to go further in vivo investigation. Methionine-choline deficiency (MCD) induced C57BL/6J NASH model mice was used for the in vivo biological experiments and the compound 3d demostrated lower liver toxicity than that of GFT 505 in the body at the same dose, and it did more effectively improve hyperlipidemia, liver fat degeneration and liver inflammation as well as significantly enhance the content of the GSH which is inportant for the liver protection. This study suggested that the compound 3d is a very promising lead compound for the treatment of NASH.

Development and application of a rapid and sensitive LC-MS/MS method for simultaneous quantification of six diterpene lactones in rats after oral administration of Rhizoma Dioscoreae Bulbiferae extract.

Diterpene lactones have been considered as the main therapeutic and hepatotoxic constituents of Rhizoma Dioscoreae Bulbiferae in recent years. In this work, a simple, rapid and accurate LC-MS/MS method was established and validated to determine six diterpene lactones in rat plasma simultaneously, including Diosbulbin B (DIOB), Diosbulbin C (DIOC), Diosbulbin D (DIOD), Diosbulbin G (DIOG), Diosbulbin J (DIOJ) and Diosbulbin L (DIOL), after oral administration of Rhizoma Dioscoreae Bulbiferae extract. The six diterpene lactones, with the inclusion of two pairs of isomer (DIOB & D, DIOC & L), and Buspirone (internal standard, IS) were successfully separated using an XDB-C18 column with the gradient elution, consisting of water with 0.1% (v/v) FA and methanol with 0.1% (v/v) FA, under a flow rate of 0.50 mL/min in 5.8 min. Precursor-product ion transitions were optimized to be m/z 362.1 → 317.1, 363.1 → 207.1, 345.0 → 299.2, 364.3 → 347.0, 396.3 → 379.3, 363.2 → 345.1 and 386.3 → 122.2 for DIOB, DIOC, DIOD, DIOG, DIOJ, DIOL and buspirone at positive ion mode with an electrospray ionization source (ESI), respectively. The linearity ranges of this present method were 0.50 to 500 µg/L for DIOB, 20.0 to 20,000 µg/L for DIOC and 2.00 to 2000 µg/L for DIOD, DIOG, DIOJ and DIOL, respectively. And the LLOQs were as low as 0.20 µg/L for DIOB, 20.0 µg/L for DIOC and 2.00 µg/L for DIOB, D, G, J and L. The accuracy of each analyte was within the range of 95.8% to 101.0% and the precision was <11.3%. No matrix effect and carry over was observed, and the recovery of the six analytes ranged from 87.3% to 109% with the RSD <11.4% within the concentrations range. The validated method was further applied to the pharmacokinetics investigation of DIOB, DIOC, DIOD, DIOG, DIOJ and DIOL successfully after oral administration of Rhizoma Dioscoreae Bulbiferae extract at 1.53 g/kg in rats.

A new strategy for choosing "Q-markers" via network pharmacology, application to the quality control of a Chinese medical preparation.

Due to its chemical complexity, proper quality control for a Chinese medical preparation (CMP) has been a great challenge. Choosing the appropriate quality markers (Q-markers) for quality control of CMP is an important work. Best of all, the chosen Q-markers are the main chemical compounds from the herbals as well as the active constituents of this CMP. Only in this way the established quality control system can really achieve the purpose of controlling the quality of CMP and ensuring the safely and effectively use of CMP. To achieve the purpose, network pharmacology combined with the contents of chemical compounds in the CMP has been used in this research. We took an anti-arrhythmic CMP, Shenxian-Shengmai oral liquid (SSOL), as an example. Firstly, UPLC-QTOF-MS/MS method was used to analyze the main components of SSOL. A total of 64 compounds were unambiguously or tentatively identified and 32 of them were further validated by reference compounds. Secondly, the network was constructed based on the identified compounds to predict the effective compounds related to cardiac arrhythmias. Based on the existing database and the operation method of topology, a method of double network analysis (DNAA) was proposed, from which 10 important targets in the pathway of arrhythmia were screened out, and 26 compounds had good antiarrhythmic activity. Based on the prediction results of network pharmacology along with the contents of the compounds in this CMP, ten representative compounds were chosen as the Q-markers for the quality control of SSOL. We find that five of these ten compounds, including danshensu, rosmarinic acid, salvianolic acid A, epimedin A and icariin, have antiarrhythmic activity. Then, the UPLC-DAD method was established as the control method for SSOL.

Correlation of TLR2 and TLR4 expressions in peripheral blood mononuclear cells to Th1- and Th2-type immune responses in children with henoch-schönlein purpura.

We discussed the correlation of TLR2 (Toll-like receptor) and TLR4 expressions in peripheral blood mononuclear cells (PBMCs) to Th1- and Th2-type immune responses in children with Henoch-Schönlein Purpura (HSP). The role of TLR2 and TLR4 in the pathogenesis of HSP was analyzed. Sixty-four HSP children treated at our hospital from October 2011 to November 2012 were enrolled and divided into NHSPN group (complicated by renal impairment, 36 cases) and HSPN group (not complicated by renal impairment, 28 cases). In the meantime, 30 normal children receiving physical examination at our hospital were recruited as controls. Peripheral blood T cell subgroups and TLR2 and TLR4 expressions in PBMCs were detected by using flow cytometry; relative expression levels of TLR2 and TLR4 mRNA in PBMCs by real-time quantitative fluorescence PCR, and plasma levels of IFN-γ, IL-4 and IL-6 by ELISA method. Relative expression levels of TLR2 and TLR4 mRNAs in PBMCs and TLR2 and TLR4 protein expressions in children with HSP were significantly higher than those of the controls (P<0.01). The relative expression levels of TLR2 and TLR4 mRNAs in PBMCs and TLR2 and TLR4 protein expressions in HSPN group were obviously higher than those in NHSPN group (P<0.05; P<0.01; P<0.01; P<0.01); CD3(+) T cells and CD3(+)CD4(+) T cells in HSP group were significantly decreased, while CD3(+)CD8(+) T cells and CD3(+)HLADR(+) T activated cells were considerably increased (P<0.01); The plasma levels of IL-4 and IL-6 in HSP group were significantly higher than those of the normal controls (P<0.01, P<0.01); IFN-γ level in the former was much lower than in the control group (P<0.05); IFN-γ/IL-4 ratio in the former was also lower than that in the control (P<0.01); TLR2 and TLR4 expressions in HSP group showed significantly positive correlation with the plasma levels of IL-4 and IL-6 (P<0.01, P<0.05; P<0.01, P<0.01) and significantly negative correlation with IFN-γ/IL-4 ratio (P<0.01; P<0.01). TLR2 and TLR4 activation may be involved in the pathogenesis of HSP. TLR2 and TLR4 overactivation may induce HSP-related renal impairment; Children with HSP showed T-cell disorders and Th1/Th2 imbalance. Activated TLR2 and TLR4 possibly mediate the pathogenesis of HSP by upregulating Th2-type immune responses.

Clinical significance of TLR3 and TLR4 in peripheral blood mononuclear cells from children with Henoch-Schönlein purpura nephritis.

The aim of the present study was to investigate the expression levels and clinical significance of Toll-like receptor (TLR) 3 and 4 in peripheral blood mononuclear cells (PBMCs) collected from children with Henoch-Schönlein purpura (HSP) nephritis. The randomized controlled trial was conducted between August 2011 and March 2013, and 105 children with a clinical diagnosis of HSP were enrolled in the study. According to the 24-h urinary protein measurements and the presence of renal damage, the 105 cases were divided into groups A, B and C as follows: Group A, children with HSP but without renal damage; group B, children with HSP nephritis but without proteinuria; group C, children with HSP nephritis and proteinuria. A total of 30 healthy children were enrolled in the normal control group (group N). The primary endpoints were the detection of TLR3 and 4 mRNA and protein expression levels in PBMCs by flow cytometry and quantitative polymerase chain reaction. The mRNA and protein expression levels of TLR4 in the PBMCs were significantly higher in groups A, B and C when compared with group N. In addition, the mRNA and protein expression levels of TLR4 in group C were much higher when compared with groups A and B. A positive correlation was identified between TLR4 protein expression and 24-h urinary protein levels in group C. The expression levels of TLR3 did not significantly differ among the groups. Protein and mRNA expression levels of TLR4 in PBMCs significantly increased and exhibited a positive correlation with urinary protein excretion. These results indicate that aberrant activation of TLR4 may be relevant to the development of HSP nephritis.