Qualitative and Quantitative Analysis of Five Bioactive Flavonoids in <i>Salix bordensis</i>Turcz. by HPLC-DAD and HPLC-ESI-MS

The qualitative characterization and quantitative analysis of five bioactive flavonoids in Salix bordensis Turcz. were achieved via reversed-phase high-performance liquid chromatography coupled with diode array detection and tandem mass spectrometry, by using an Agilent ZORBAX SB-C18 HPLC column with a gradient elution of 0.3% (v/v) formic acid in water and methanol as the mobile phase. The compounds in the mixture were clearly identified by comparing their HPLC-DAD ultraviolet spectra, retention times, and MS data with those of corresponding reference compounds. All calibration curves showed good linearity (r2 > 0.9998) within the test ranges. The LOD, LOQ, specificity, precision, and accuracy for the method were validated. The results demonstrated that this analytical approach is ideal for the determination of bioactive compounds, such as flavonoids, and that it constructed a basis for the comprehensive evaluation of the quality of Salix bordensis Turcz.

Potentilla anserina polysaccharide alleviates cadmium-induced oxidative stress and apoptosis of H9c2 cells by regulating the MG53-mediated RISK pathway

Oxidative stress plays a crucial role in cadmium(Cd)-induced myocardial injury.Mitsugumin 53(MG53)and its mediated reperfusion injury salvage kinase(RISK)pathway have been demonstrated to be closely related to myocardial oxidative damage.Potentilla anserina L.polysaccharide(PAP)is a polysaccharide with antioxidant capacity,which exerts protective effect on Cd-induced damage.However,it remains unknown whether PAP can prevent and treat Cd-induced cardiomyocyte damages.The present study was desgined to explore the effect of PAP on Cd-induced damage in H9c2 cells based on MG53 and the mediated RISK pathway.For in vitro evaluation,cell viability and apoptosis rate were analyzed by CCK-8 assay and flow cytometry,respectively.Furthermore,oxidative stress was assessed by 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)staining and using superoxide dismutase(SOD),catalase(CAT),and glutathione/oxidized glutathione(GSH/GSSG)kits.The mitochondrial function was measured by JC-10 staining and ATP detection assay.Western blot was performed to detect the expression of proteins related to MG53,the RISK pathway,and apoptosis.The results indicated that Cd increased the levels of reactive oxygen species(ROS)in H9c2 cells.Cd decreased the activities of SOD and CAT and the ratio of GSH/GSSG,resulting in decreases in cell viability and increases in apoptosis.Interestingly,PAP reversed Cd-induced oxidative stress and cell apoptosis.Meanwhile,Cd reduced the expression of MG53 in H9c2 clls and inhibited the RISK pathway,which was mediated by decreasing the ratio of p-Akt^(Ser473)/Akt,p-GSK3β^(Ser9)/GSK3β and p ERK1/2/ERK1/2.In addition,Cd impaired mitochondrial function,which involved a reduction in ATP content and mitochondrial membrane potential(MMP),and an increase in the ratio of Bax/Bcl-2,cytoplasmic cytochrome c/mitochondrial cytochrome c,and Cleaved-Caspase 3/Pro-Caspase 3.Importantly,PAP alleviated Cd-induced MG53 reduction,activated the RISK pathway,and reduced mitochondrial damage.Interestingly,knockdown of MG53 or inhibition of the RISK pathway attenuated the protective effect of PAP in Cd-induced H9c2 cells.In sum,PAP reduces Cd-induced damage in H9c2 cells,which is mediated by increasing MG53 expression and activating the RISK pathway.

Anti-inflammatory activities of gentiopicroside against iNOS and COX-2 targets

Objective: To isolate high-purity gentiopicroside from the Chinese herbal Gentiana officinalis and investigate its anti-inflammatory activity against iNOS and COX-2 targets.Methods: The purity and structures of gentiopicroside were determined by HPLC, IR, NMR, and MS. The anti-inflammatory effects of gentiopicroside were investigated by in vivo, in vitro, and molecular experiments.Results: In vitro experiment results showed that gentiopicroside inhibited nitric oxide(NO), prostaglandin E2(PGE2), and interleukin-6(IL-6) production in mouse macrophages RAW 264.7 stimulated by lipopolysaccharide. In vivo experiment found that xylene-induced mouse ear swelling was inhibited by gentiopicroside with an inhibition rate of 34.17%. Molecular docking of cyclooxygenase-2(COX-2) and inducible nitric oxide synthase(i NOS) with gentiopicroside showed that hydrogen bonds(H-bonds) were formed between the sugar fragments in gentiopicroside structure with Tyr355, Ser353, Leu352, Ser530,Arg120, and His90 of COX-2, and Glu377, Asp382, Tyr373, Tyr347, Gln263, Asn370, and Gly371 of i NOS.Thus, gentiopicroside had a lower docking score and displayed satisfactory anti-inflammatory activities.Conclusion: These results suggested that the mechanism of anti-inflammatory activity of gentiopicroside was associated with the downregulation of inflammatory cytokines, such as NO, PGE2, and IL-6, and the suppression of iNOS and COX-2. Therefore, gentiopicroside is a potential and selective iNOS and COX-2 inhibitor.

Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy via PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway

OBJECTIVE:To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2(PINK1/PARKIN)signaling pathway.METHODS:3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells;the hypoxia/reoxygenation(H/R)model was established in tri-gas incubator;2’,7’-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species(ROS);the changes of mitochondrial membrane potential was determined by 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide staining;the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits;flow cytometry was used to detect the ratio of apoptotic cells;transmission electron microscope was used to observe the ultrastructure of H9C2 cells;Western blot was used to detect the protein changes of mitochondrial 20 k Da outer membrane protein(TOM20),translocase of inner mitochondrial membrane 23(TIM23),presenilins associated rhomboid-like protein(PARL),PINK1,PARKIN and mitofusin 1(Mfn1),mitofusin 2(Mfn2),phosphotyrosine independent ligand for the Lck SH2 domain of 62 k Da(P62),microtubule-associated protein 1 light chain 3 beta(LC3B);the m RNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction;immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin.RESULTS:Resveratrol could inhibit the proliferation of H9C2 cells in a time-and concentration-dependent manner;however,pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels,alleviate the loss of mitochondrial membrane potential induced by H/R,inhibit H/R-induced apoptosis of H9C2 cells,and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage.Resveratrol could further increase the levels of p62,PINK1,PARKIN protein,the expression of PINK1,PARKIN m RNA and the ratio of LC3BⅡ/LC3BⅠin H/Rinduced H9C2 cells,inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells,and further reduce the expression of TOM20,TIM23,PARL,Mfn1 and Mfn2 protein in H/R-induced H9C2 cells.The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells.CONCLUSIONS:Resveratrol can protect H9C2 cells from H/R injury,which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.