Effects Of Fomes Officinalis Ames Polysaccharides Against Mitochondrial Dysfunction And Oxidant Stress In Aβ-treated PC12 Cells And AD Mice

Objective:This study aimed to investigate the protective mechanism of Fomes officinais Ames polysaccharides components(FOPSa)and(FOPSb)against mitochondrial damage and oxidative stress in Alzheimer’s disease(AD)mices and Aβ25-35-induced PC12 cells.Methods:1.Aβ25–35 was used to induce mitochondrial damage and oxidative stress in PC12 cells.FOPS components(FOPSa and FOPSb)with different concentrations were incubated with PC12 cells for 2h,followed by administration with β-amyloid protein fragment 25–35(Aβ25–35).Then PC12 cells were divided control group,Donepezil hydrochloride(DHCL)group,model group,FOPSa group and FOPSb group.Cell apoptosis was measured using an Annexin-V-FITC apoptosis detection kit.Cellular ATP concentration,superoxide dismutase(SOD),malondialdehyde(MDA),lactate dehydrogenase(LDH)and mitochondrial membrane potential(MMP)were determined by commercial kits.Cytochrome C in the mitochondria and cytosol was determined by Western blot analysis.PC12 cells were cultured with Aβ25-35 to establish AD cell model and were randomly divided into control group,Aβ25-35(40μmol/L)group,Aβ25-35+FOPSa groups and Aβ25-35+FOPSb groups.Targeting probes were used to track the subcellular localization of Aβ25-35.The changes of reactive oxygen species(ROS)in PC12 cells were detected by the commercial kit.Western blot was used to analyze the expression of apoptosis-related proteins(Bax and Bcl-2),Nrf2,apoptosis signal-regulating kinase 1(ASK1)and phosphorylated ASK1 proteins.2.Sixty-four 3-month-old APP/PS1 double-transgenic male mice were randomly divided into 8 categories,with 8 3-month-old C57BL/6 mice as the control group subjected to continuous intragastric administration for 90 days.Morris water maze test was performed to examine the behavior changes,colorimetric method to detect activity changes of SOD,glutathione peroxidase(GSH-Px)and ATP,together with the content changes in MDA.Congo red staining method was adopted to track the changes of the brain neurons of mice as well as brain tissue.Western blot were conducted to observe the content changes of each group in Camp response element-binding protein(CREB),and peroxisome proliferators activated receptor gamma co-activator 1 alpha(PGC-1α).3.PC12 cells were divided into the control group,model group,FOPSa and FOPSb groups(200 μg/m L).Whole transcriptome sequencing was applied to screen long noncoding RNAs(lnc RNAs)and m RNAs with differential expression among the control,model,FOPSa and FOPSb groups.GO and KEGG databases were applied to analyze the function and signal pathway of significant expression genes involved in regulating mitochondrial damage and oxidative stress.Results:1.FOPSa and FOPSb could significantly decrease LDH release,MDA level but increase the accumulation of SOD induced by Aβ25–35 in PC12 cells in a dose-dependent manner.They could also effectively prevent Aβ25–35-stimulated cytotoxicity,which involved in attenuating cell apoptosis,and inhibiting Cytochrome C release from mitochondria to cytosol in PC12 cells.Moreover,FOPSa and FOPSb significantly alleviated mitochondrial dysfunction by regulating the MMP,as well as promoting the mitochondrial ATP synthesis.It was found that mitochondrial integrity was damaged by Aβ25-35,while the mitochondrial damage was alleviated after pretreatment with 200μg/m L FOPSa or 200μg/m L FOPSb.FOPSa and FOPSb could significantly inhibit the accumulation of ROS induced by Aβ25-35 in PC12 cells in a dose-dependent manner.They could also effectively prevent Aβ25-35-stimulated cytotoxicity,which involved in attenuating cell apoptosis,increasing Nrf2 expression and the ratio of Bcl-2/Bax,as well as inhibiting ASK1 and phosphorylated ASK1 proteins levels.2.Compared to the model group: In the water maze test,an increase in the activity of mice was detected in FOPSa and FOPSb treatment groups,with various degrees of recovery in their learning and memory ability.It is also worth mentioning that the activities of SOD,GSH-Px and ATP showed an increase,while the content of MDA featured a decrease.The results from Congo red staining showed that an obvious Aβ accumulation was detected in the brain tissues of the model mice group.Western blot analysis showed a decrease in the contents of CREB and PGC-1α.However,the accumulation of Aβ in the FOPSa and FOPSb groups marked a drop in both its number and volume;the contents of CREB and PGC-1α increased.3.GO analysis suggested that FOPSa and FOPSb intervention changed the levels of lnc RNAs and m RNAs.At the m RNA level,identified 11 validated entries associated with mitochondrial and oxidative stress were identified,including mitochondrial transport,mitochondrial respiratory chain complex II,mitochondrial envelope and response to oxidative stress regulation.At the Lnc RNA level,a valid entry related to mitochondrial and oxidative stress was notifiedied,which was involved in the regulation of transcription from RNA polymerase II promoter.Conclusion:1.FOPSa and FOPSb played neuroprotective roles against Aβ25–35-induced cytotoxicity in PC12 cells through suppressing the mitochondrial apoptosis pathway.The mechanism may be related to its suppression of the activation of Nrf2 signal pathway.2.FOPSa and FOPSb can significantly improve the learning and memory ability of AD mice,inhibit the production and accumulation of Aβ,and exert protective effects on the neurons of AD mice by improving the mitochondrial function and stimulating oxidative stress pathway.3.FOPSa and FOPSb play an antidepression effect through the regulation of multiple genes and signal pathways,including apoptosis,mitochondrial dysfunction and oxidative stress pathways.