Resveratrol-induced Mitophagy Attenuates Oxidative Damage In Macrophages

Resvratrol (Resv), a natural product, is an antioxidant, exhibiting obvious therapeuticeffects on many diseases. However, the mechanism studies were still lingered on thestage of scavenging reactive oxygen species or by interacting with antioxidantenzymes directly. We had found in preliminary experiments that autophagy wasrelated to antioxidant and autophagy induced by Resv. In this study, the role of Resvinduced mitophagy and the involved signaling transduction was further studied.Kun Ming (KM) mice loaded with restraint stress were induced by fixed in therestraint cage to establish a oxidation stress model. And they were randomly dividedinto normal group, restraint stress group, Resv low-dose group (i.g.7.5mg/kg), Resvmiddle-dose group (i.g.15mg/kg), Resv high-dose group (i.g.30mg/kg), rapamycin(Rapa) group and Vitamin C (VitC) group, with6mice each. After restraint stress for18h and free for the following12h, we assessed the effects of Resv on oxidativedamage of mouse peritoneal macrophages induced by restraint stress. It was foundthat Resv as well as antioxidant VitC mitigated restraint stress induced mitochondrialdysfunction, apoptotic death and the decline of phagocytosis in mouse peritonealmacrophages. Autophagy agonist rapamycin (Rapa) attenuated the oxidative damagesdescribed above. Meanwhile, a compensatory increase of autophagysomes induced byrestraint stress were seen, which was enhanced by Resv administration.We discuss the mechanism of relieving the oxidative damage by Resv on RAW264.7macrophages, using a oxidative stress model triggered by AAPH (5mM,24h).Several experiments were performed.(1) Inhibition ratio of macrophages proliferationwas determined by MTT and apoptotic bodies were observed by microscopy. Inaddition, flow cytometry was used to determine phagocytic rate. Experimental resultsshowed that AAPH inhibited cell growth in concentration and time dependent manner.Typical apoptotic deaths in cells induced by AAPH were shown in Morphologicaldata. Moreover, further experiment showed that inhibition ratio of macrophages proliferation by AAPH was attenuated by Resv. Meanwhile, the decline ofphagocytosis was reversed by Resv.(2) Intracellular ROS content was determined byfluorescence spectrophotometer and mitochondrial membrane potential (MMP) wasexamed by flow cytometry. In addition, the expression level of mRNA NDUFS8wasalso detected by RT-PCR. Morphology and RT-PCR results showed that Resv rescuedmitochondrial dysfunction and apoptotic death. The effects of Resv were augmentedby rapamycin while inhibited by chloroquine/3-MA, suggesting that autophagy wasinvolved.(3) Expressions of autophagy related proteins (Beclin1and LC3β) andproteins involved in SIRT3/AMPK signaling pathwaywere determined by Westernblotting. Transmission electron microscopy and confocal microscopy were applied forobservation of typical autolysosomes. Confocal microscopy and Western blottingconfirmed the role of autophagy. Further transmission electron microscopy showedvisible mitophagy induced by Resv, indicating a recycle of damaged mitochondria.(4)RNA interference of SIRT3was utilized to demonstrate the role ofSIRT3/AMPK/autophagy signaling pathway involved in the protective effect of Resv.Subsequent experiments showed autophagy positively regulated SIRT3(amitochondrial sirtuin) and AMP-activated protein kinase (AMPK), while siRNA ofSIRT3downregulated activation of AMPK and induction of autophagy, suggesting apositive regulatory feedback. Moreover, interference of SIRT3partly decreasedpositive effect of Resv on phagocytosis. Vitamin C and edaravone, as the antioxidantcontrols, showed no effects upon induction of autophagy. We demonstrate that, apartfrom antioxidation, Resv mitigated oxidative stress and recovered phagocytosis inmacrophages was partly through SIRT3/AMPK/autophagy positive feedback.In conclusion, restraint stress or the application of AAPH could induce theproduction of ROS, which lead to functional damages in mitochondria. Oxidativestress in mitochondria could cause a compensatory up-regulation of SIRT3, activatingAMPK pathway and consequently the induction of autophagy. However, this kind ofcompensatory autophagy was not enough to ameliorate oxidative damages within thecell. The administration of Resv could enhance the level of autophagy, which was alsofound to be related to the SIRT3/AMPK pathway. Further mechanism research proved that Resv protects macrophages against oxidative stress was partly through theSIRT3/AMPK/autophagy positive feedback. The current study elucidated themechanism of mitophagy in the amelioration of oxidative stress, which provided anovel presective on antioxidation mechanism and mitophagy signaling pathway. Thedata obtained in this study would benefit the research of mitochondria quality controlin different diseases.