1.Structural identification of the related substances of fusidic acid by LC-MS
Fangmei LIANG ; Yueling NI ; Lu WANG ; Taijun HANG ; Min SONG
Journal of China Pharmaceutical University 2018;49(3):322-332
To identify the related substances of fusidic acid by LC-MS,separation was performed on an Agilent Extend-C18column(150 mm ×4. 6 mm,3. 5 μm)by linear gradient elution with a mobile phase consisting of methanol,acetonitrile and formic acid. Electrospray positive ionization high resolution TOF/MS was used for the determination of the accurate mass and elemental composition of parent ions of the related substances;triple qua-drupole tandem mass was employed for the mass spectra determination of the product. The structures of the related substances were then figured out through the elucidation of the fragment ions. Fusidic acid and its related sub-stances were adequately separated under the established HPLC conditions. Nineteen major related substances of fusidic acid were detected and speculated by hyphenated techniques. Eleven of them were recorded in European Pharmacopoeia,while the others have not been previously reported. The established LC-MS method is effective for the separation and identification of the related substances of fusidic acid and the results are useful for its storage conditions and quality assurance.
2.The mechanism of resveratrol promoting FNDC5 degradation in skeletal muscle of male obese mice
Fangmei Yu ; Qiongqiong Cao ; Dongmei Xu ; Yueming Long ; Heng Zhou ; Li Gui ; Yunxia Lu
Acta Universitatis Medicinalis Anhui 2023;58(3):412-417
Objective:
To investigate the mechanism of resveratrol promoting fibronectin type Ⅲ domain-containing 5 (FNDC5) degradation in skeletal muscle of male obese mice.
Methods:
Six-week-old male C57BL /6 mice were randomly divided into three groups : standard control diet ( SCD) ,high-fat diet ( HFD) and high-fat diet treated with resveratrol (HFD + RES) .HFD + RES group was intervened with resveratrol via gavage [400 mg / kg · d) ] while fed HFD for 20 weeks.The body mass,serum TG,TC,LDL-C and HDL-C levels were detected.The pathological changes in skeletal muscle were detected by HE staining.The expression of FNDC5,SIRT1,SIRT2,LC3, p62,Beclin-1,ATG5,ATG7 was assessed by immunohistochemistry,RT-PCR and Western blot respectively.
Results:
The body mass ,serum TG ,TC and LDL-C levels increased significantly ,meanwhile HDL-C levels decreased in HFD group.Lipid deposition between skeletal muscle fibers were obvious in HFD group.The immuno- histochemistry results showed that protein expression levels of SIRT1,SIRT2 and LC3 obviously decreased,while the protein levels of FNDC5 and p62 obviously increased.The expression levels of FNDC5 significantly increased, while the gene expression levels of SIRT1,SIRT2,LC3,Atg7 and Beclin-1 obviously decreased.All these responses were attenuated by treatment with RES.
Conclusion
RES has obvious effects of lipid-lowering and promoting FNDC5 degradation in skeletal muscle tissues,which may be related with SIRT1 and SIRT2-induced autophagy, thus resulting in degradation of FNDC5 .
3.Oxidative stress injury and transcriptome analysis of human myocardial AC16 cells induced by zinc oxide nanoparticles
Fangmei Lu ; Yujie Li ; Jiabin Guo ; Dexiang Xu ; Chao Liu
Acta Universitatis Medicinalis Anhui 2023;58(3):450-456
Objective:
To investigate the oxidative stress injury of nano zinc oxide nanoparticles(ZnO NPs) on human myocardial cells (AC16) ,and to analyze the mechanism of ZnO NPs from the transcriptome level.
Methods:
Dynamic light scattering (DLS) was used to characterize and detect ZnO NPs.After AC16 cells were exposed to ZnO NPs at different doses and at different times,the cell survival rate was determined by CCK-8 method.AC16 cells were divided into control group,ZnO NPs (50,100,200 μmol /L) ,after 6 h treatment,the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were measured.AC16 cells were divided into control group,50 μmol /L ZnO NPs group and 200 μmol /L ZnO NPs group.After 6 h exposure,total RNA was extracted by TRIzol for transcriptome analysis ,and the differentially expressed genes were enriched by gene body ( GO) ,Kyoto Encyclopedia of Genes and Genomes (KEGG) .
Results :
The results of DLS showed that the hydrodynamic diameter was ( 192. 2 ± 1. 63 ) nm and the Zeta potential was ( -23. 26 ± 1. 05 ) mV. CCK-8 results showed that the survival rate of AC16 cells decreased with the increase of dose and time of exposure to ZnO NPs. Fluorescence quantification showed that with the increase of ZnO NPs exposure dose,MMP significantly decreased at 100 μmol /L ZnO NPs(P<0. 05) ,and ROS significantly increased at 50 μmol /L ZnO NPs(P<0. 05) .Using the multifunctional microplate reader,it was observed that MMP and ROS were statistically significant at 100 and 50 μmol /L ZnO NPs,respectively,showing a decrease in MMP and an increase in ROS.Transcriptome analysis showed that 1 071 genes were enriched in the 50 μmol /L ZnO NPs group compared with the control group,including 561 up-regulated genes and 510 down-regulated genes.Compared with the control group,7 164 genes were enriched in 200 μmol /L ZnO NPs group,including 4 098 up-regulated genes and 3 066 down-regulated genes.GO and KEGG analysis showed that the differential genes were mainly concentrated in ROS,antioxidant activity,mitochondrial cytochrome C release,apoptosis and other signaling pathways.
Conclusion
ZnO NPs can decrease the survival rate of AC16 cells and induce mitochondrial damage and oxidative stress,among which ROS-mediated oxi- dative stress and mitochondrial function changes are important toxic mechanisms of ZnO NPs induced AC16 cytotoxicity.