| Platycodon grandiflorus(Jacq.)A.DC.(PG),the dry root of the platycodon grandiflorum herbaceous plant,which has the effect of promoting lung phlegm,pharynx and purulent swelling.Its chemical components are polysaccharide,saponins,flavonoids,fatty oils,fatty acids,etc.Modern pharmacological studies have shown that PG has anti-cancer,anti-oxidation,immune regulation,anti-inflammatory and anti-bacterial,anti-tumor,hypolipidemic,hypoglycemic,liver-protecting,anti-lung injury and other effects.Platycodon grandiflorus polysaccharide(PGPS)is one of the biologically active components of PG,which has anti-oxidation,immune enhancement and anti-tumor effects.Carbonyl cyanide 3-chlorophenylhydrazine(CCCP)is a proton carrier,mitochondrial oxidative phosphorylation uncoupling agent that promotes the permeability of mitochondrial inner membrane to H~+,leading to loss of membrane potential on both sides of the mitochondrial inner membrane,induced apoptosis.Mitochondria are the regulatory centers of apoptosis,play an important role in the process of apoptosis.The purpose of this study was to investigate the potential protective effect of total PGPS_t on CCCP-induced mitochondrial apoptotic pathway in 3D4/21 cells.The cell viability was determined by CCK-8 method.The results showed that the survival rate of 15μM CCCP-treated cells was 51.4%.The cell viability of PGPS_t(100μg/mL,200μg/mL)and CCCP co-treatment was 62.2%and 78.6%.Which indicated that PGPS_t could inhibit cell damage.Cells were treated with Annexin V and PI double staining,and apoptosis rate was detected by flow cytometry.The results showed that the apoptosis rate of cells treated with 15μM CCCP for 12 h was 34.7%,using PGPS_t(100μg/mL,200μg/mL)and CCCP co-treatment,and the apoptosis rate decreased to 29.8%,27.2%,which indicated that apoptosis was inhibited.The cells were stained by Hoechst 33342 method,the morphology of apoptotic cells was observed by laser confocal microscopy.The results showed that 15μM CCCP treated 12 h,the nucleus was deeply stained,invagination and nucleus shape were irregular.PGPS_t(100μg/mL,200μg/mL)was co-treated with CCCP,the phenomenon of nuclear deformation was significantly reduced,indicating that PGPS_t has protective effect on apoptotic cells.The change of mitochondrial membrane potential was detected by JC-1 method.The mitochondrial membrane potential decreased significantly after treatment with 15μM CCCP for 12 h,indicating that mitochondrial damage was caused by CCCP treatment.PGPS_t(100μg/mL,200μg/mL)co-treated with CCCP inhibited the decrease of membrane potential,indicating that PGPS_t can inhibit the change of mitochondrial membrane potential.Expression of apoptosis-related proteins Caspase-9,Cleaved Caspase-3and Bcl-2 detected by Western Blot.The results showed that the expression of Caspase-9 and anti-apoptotic protein Bcl-2 protein decreased and the expression of Cleaved Caspase-3 protein increased after 15μM CCCP treated cells for 12 h,which indicated that CCCP induced apoptosis.PGPS_t(100μg/mL,200μg/mL)treated cells,Caspase-9 and anti-apoptotic protein Bcl-2 protein expression increased,Cleaved Caspase-3 protein expression decreased,indicating that PGPS_t significantly inhibited CCCP-induced apoptotic protein changes.In conclusion,PGPS_t can antagonize CCCP-induced apoptosis of 3D4/21 cells by restoring mitochondrial membrane potential,protecting nuclear morphology and increasing the expression of anti-apoptotic protein Bcl-2,the effect was significant at 200μg/mL. |
