Saponins From Panax Japonicus Attenuated Neuronal Mitochondrial Injury Of Aging Rats

Background Currently,aging and related neurodegenerative diseases have become one of the most serious diseases affecting the health of the elderly,and neuronal mitochondrial dysfunction is one of the major pathological manifestations of aging and related neurodegenerative diseases.Objective To investigate the protective effect of Saponins from Panax Japonicus(SPJ)on neuronal mitochondrial injury of aging rats and the underlying mechanism based on the mitochondrial dynamics pathway.Methods Experiment 1: Different ages of SPF-rated SD male rats were divided into the Young(5M)group,Aging group(22M),SPJ low-dose group(10 mg/kg,22M)and SPJ high-dose group(30 mg/kg,22M).The rats in the drug intervention group were treated from18 months old for consecutive 4 months until they were 22-month-old.After weighing,the rats were anesthetized with uratan for perfusion or execution.Brain tissue from the perfused rats were used to prepare paraffin sections,while the cortex and hippocampus from the unperfued rats were collected and used to assay.(1)Nissl staining was used to observe the neuronal damage;(2)Neuronal morphology,synaptic density and mitochondrial structure in the cortical region were observed by transmission electron microscopy;(3)The protein expression of mitochondrial fission(Drp1)and mitochondrial fusion(Mfn2,Opa1)in the hippocampus were detected by western blot.Experiment 2: The SH-SY5 Y cells were divided into normal control group,D-Galactose(D-Gal)model group and D-Gal + SPJ(25 μg/m L,50 μg/m L)group.After incubated with different concentrations of SPJ for 12 h,SH-SY5 Y cells were stimulated with 200 m M D-Gal for 48 h to establish the cell senescence model and to evaluate the following indexes :(1)Cells viability was detected by MTT assay;(2)The morphological changes of SH-SY5 Y cells was observed by light microscopy;(3)The cell senescence was observed by β-galactosidase(β-gal)staining;(4)The mitochondrial ultrastructure of SH-SY5 Y cells was observed by transmission electron microscopy;(5)ROS level of SH-SY5 Y cells was detected by fluorescence probe DCFH-DA;(6)Mitochondrial membrane potential of SH-SY5 Y cells was detected by fluorescent probe JC-1;(7)ATP levelof SH-SY5 Y cells was detected by phosphomolybdic acid colorimetric assay;(8)The protein expression of mitochondrial fission(Drp1)and mitochondrial fusion(Mfn2,Opa1)of SH-SY5 Y cells were detected by western blot.Results Experiment 1: Compared to the young group,the quantity of neuronal Nissl body in the cortex and hippocampus of the aging group became less(P < 0.01),the cortical neurons were pyknotic,the nuclear membrane was shrunk,and the nucleoli were shifted,whereas the SPJ treatment improved all the above neuronal damage;The synaptic density in the cortex of aging rats was reduced,the mitochondrial cristae was broken,and the aspect ratio and area of mitochondria were decreased(P < 0.01),SPJ treatment increased synaptic density,mitochondrial aspect ratio and area(P < 0.05);The expression of mitochondrial fission protein(Drp1)increased(P < 0.01),and the expression of fusion proteins(Mfn2,Opa1)decreased(P < 0.05 or P < 0.01)in the hippocampus of aging rats.SPJ treatment reduced the expression of mitochondrial fission protein(Drp1)(P < 0.05 or P < 0.01)and increased the expression of fusion proteins(Mfn2,Opa1)(P < 0.05 or P < 0.01).Experiment 2: Compared to the control group,cell viability decreased and β-gal staining increased significantly after stimulation with 200 m M D-Gal(P < 0.01),which can be reversed by SPJ treatment at the doses of 25 μg/m L and 50 μg/m L(P < 0.01).Meanwhile,D-Gal stimulation led to mitochondrial cristae disorderly arranged and even disappeared and decreased the aspect ratio and area of mitochondria(P < 0.01),whereas SPJ treatment improved the mitochondrial cristae structure,increased the number of tubular mitochondria,and enhanced the mitochondrial area and aspect ratio caused by the D-Gal stimulation(P <0.05 or P < 0.01).Furthermore,D-Gal stimulation enhanced the ROS level and declined the mitochondrial membrane potential and ATP level of SH-SY5 Y cells(P < 0.01),while SPJ treatment alleviated the ROS level(P < 0.01)and increased the mitochondrial membrane potential and ATP level(P < 0.05 or P < 0.01).The further study demonstrated that D-Gal stimulation upregulated the expression of mitochondrial fission protein(Drp1)and downregulated the expression of mitochondrial fusion protein(Mfn2,Opa1)of SH-SY5 Y cells,however,SPJ treatment decreased the expression of Drp1(P < 0.01)and enhanced the expression of Mfn2 and Opa1(P < 0.05 or P < 0.01).Conclusion SPJ improved the neuronal mitochondrial injury of aging rats through regulate the mitochondrial fission-fusion homeostasis.