3.1 HDAC6 and prx
Peroxiredoxin (Prx)is one of the proteins that maintains intracellular redox homeostasis, to function as protection of the cell against injury by radical accumulation; it catalyzes the reduction of peroxidatives to protect cell from oxidative stress(Park et al., 2016, Ledgerwood et al., 2017). The Prx family contains 6 anti-oxidative proteins, Prx 1–6 (Hu, et al., 2011). Prx possesses multiple functions. In addition to involving regulation of cell process, including growth, metabolism, cell death and surviving (Leyk et al., 2017), it acts as sensor of redox reaction; its peroxidation and inactivation may be resulted from oxidative stress, to reduce the cellular anti-oxidative potential(Rhee et al., 2012) .
Available data suggests that HDAC6 modulates activity of Prx through acetylation modification to regulate oxidative injury.
The pretreatment of HDAC6 inhibitor Tubastatin A stimulates induction of Prx1 activity in a dose dependent manner in H2O2 challenged 661W cells,to restore the H2O2 mediated injury(Leyk, et al., 2017) . In Parkinson syndrome model mice induced by 6-OHDA, the pharmacologic inhibition of Tubastatin A induces the acetylation level of Prx1 and Prx2 in dopamine energistic neurons,and decreases the production of ROS to protect dopamine energistic neuron from 6-OHDA induced death( Jian et al. , 2017) .
Further study suggests that, reduction activity of Prx1 on peroxidase is dependent on acetylation mediated by HDAC6 (Parmigiani et al., 2008, Ledgerwood et al., 2017) . In primary hippocampus cells and HT22cells,acetylation of Prx1 is reduced in model of Alzheimer disease (AD) model, and restored by Tubastatin A. The change of ROS is correlated to acetylation of Prx1 but independent of the amount of Prx1. The data suggested that level of acetylation increase shows more potential anti-oxidative and reduction activity. As further investigation has shown that the mimic mutant K197Q transfectant HT22 cells partially resist elevation of ROS in AD model, while acetylation silencing mutant presents similar result as the group of wild type (WT).
In silencing mutant Prx1 transfectant HT22 cells, co-incubation of Tubastatin A and Aβ does not restore the level of ROS, but in of wild type Prx1. The findings suggest that inhibition of HDAC6 reduces the level of ROS through deacetylation of K197 on Prx1 (Choi et al. , 2017) . In addition, Ca2+ presents identical change with ROS, and with Ca2+inhibitor BAPTA-AM reduces Aβ mediated ROS elevation,suggesting that reduction of ROS caused by Prx1 acetylation is partially attributed to the reduction of Ca2+. Meanwhile, similar findings are obtained from in vivo experiments. In comparison with wild type control, AD model of 5xFAD mice show reduced Prx1 acetylation in brain tissues, but injection HDAC6 inhibitor reverses the reduction, and reduces oxidative stress (Choi et al. , 2017). Similar observations have been recorded in ischemia-reperfusion rat and myocardiocytes (Leng et al., 2018) .
3.2 HDAC6 and mitochondria
In most mammalian cells, mitochondria supply energy for cells using ATP generated in phosphorylation-oxidation utilizing O2 as ultimate electron receptor[78; Rastogi et al., 2006)]. ROS is produced in the process (Hwang et al., 2016) . The main ROS generated by mitochondria is O2−. A majority of O2− degrades to form H2O2 (Murphy, 2009). Research suggests that production of ROS is the indicator to evaluate mitochondrial functions ( Ramalho-Santos et al., 2009).
Under normal condition, ROS is controlled at normal level through anti-oxidative system in cells. Over-produced ROS induces oxidative stress, and oxygenates mitochondrial membrane proteins, altering permeability of the outer membrane. Disturbance mitochondrial membrane potential leads to the release cytochrome c, triggering apoptosis (Petrosillo et al., 2003) . The over-production of ROS causes mitochondrial dysfunction, and oxidative damage and in turn increases the production of ROS. The adverse circulation feed-forward loop promotes the disease progression (El-Amine et al., 2018) . HDAC6 activity contributes to mitochondrial dysfunction, and hence oxidative stress (Leng et al., 2018, Bai et al., 2015) .
HDAC6 has been known to modulate activities of mitochondria in many types of living cells. For example, HDAC6 may cause the loss of ability to eliminate damaged mitochondria by autophagy (Lee et al., 2010) . In RAW264.7 cells pretreated with HDAC6 inhibitor ACY-1215, the mitochondrial ultrastructure and mitochondrial membrane activated by lipopolysaccharide (LPS)restore to normal (Zhang et al. 2019a).
Investigations indicate that HDAC6 controls oxidative stress injury through modulation of mitochondria. Superoxydites and endogenous thioredoxin1 (Trx1) derived from mitochondria are elevated in HuREC cells (human retinal endothelial cells)cultured in hyperglycose medium and the negative effect is inhibited by Tubastatin A( Abouhish et al., 2020) . Proteomic data reveals a remarkable decrease of mitochondrial inner membrane and protein complex in acute liver failure (ALF) model of mice, while specific inhibitor of HDAC6, ACY-1215 treatment reverses such change, production of ROS reduces in ALF mice,together with reduction of liver cell necrosis; the effect is achieved by normalizing the activity of mitochondrial electronic transmission chain complex I and restoring oxidation-phosphorylation in ALF mice (Zhang, et al., 2019b).
PHB1(Prohibitin 1)is a protein essential for the properly functioning of mitochondria. Data indicates that in pyrotoxicemic patient derived peripheral blood mononuclear cells (PBMCs) and pyrotoxicemic rat, its expression level is negatively correlated with HDAC6, and inhibition of HDAC6 ameliorates injury due to mitochondrial damage and pyrotoxicemia. It is therefore speculated that inhibition of mitochondrial by PHB1cures the injury caused by pyrotoxicemia through HDAC6 regulation (Guo et al. , 2020) .
Other study reveals that HDAC6 modulates mitochondrial function through ROS. In A375.S2 cells, HDAC6 inhibition by siRNA results in elevation of ROS, drop of mitochondrial membrane potential, the reduction of PGC-1, the key regulatory elementof mitochondrial biogenesis, elevation of mitochondrial fusion protein Mfn2and reduction of mitochondrial division protein DRP1. The changes are restored by ROS inhibitor. Mitochondrial fusion and division are the key process for continuous remodeling network dynamics of mitochondria, suggests that HDAC6 modulates mitochondrial function through ROS ( Bai, et al., 2015).
Contracting results have been obtained from different research. Evidence directly supports that in human melanoma cells highly expressing HDAC6, knockdown of triggers remarkable production of ROS and drop of mitochondrial membrane potential ( Bai, et al., 2015). In A549 cells, knockdown of HDAC6 reduces mitochondrial enzymatic activities (Kamemura et al., 2012). Other study suggests that in mesenchymal stem cell (MSC) the knockdown of HDAC6 elevates level of ROS. It may be explained by disturbance of oxidative metabolism of mitochodnria (Park et al., 2017) . The HDAC6 knockout mice present no phenotype related to mitochondrial defect (Gao et al., 2007, Zhang et al., 2008; Lee et al., 2010). This indicates that HDAC6 exerts effect to maintain intact of mitochondria during stress condition (Lee et al., 2014).
3.3 HDAC6 and autophagy
Autophagy is a lysosome dependent process to degrade metabolites, during which dysfunctional organelles and misfolded proteins and radical oxygen are reused to maintain cellular homeostasis ( Parzych K, Klionsky , 2014). It is an efficient adaptation process, which protects cells from injury caused by various stimuli, including by lysosome, degradation of dysfunctional organelle and protein clusters, by oxidative stress (Lavandero et al, 2013; Chen and Klionsky, 2011). When the organelles and proteins are engulfed in the autophagosome, and then it is fused with lysosomes, and the target substances are hydrolyzed by enzymes from lysosome (Mizushima and Komatsu , 2011; Levine and Kroemer, 2008) . Meanwhile, autophagy is a key cellular event to antagonize mitochondrial damage mediated by anti-oxidative stress (Zhu et al., 2013). In mammalian cells, the accumulation of mitochondrial ROS and lipid oxidation plays an essential role in authophagy, as both O2− and H2O2 trigger autophagy (Scherz-Shouval, et al., 2007). Autophagy plays an important role in maintaining normal oxidation and reduction equilibrium through degradation of substances produced by injured cells (Lavandero et al, 2013; Chen and Klionsky, 2011).
The link between HDAC6 and autophagy has been demonstrated; but different results present in the specific modulation in different study. Some data suggests that inhibition of HDAC6 reduces autophagy. Through recruitment of epidermal actin dependent actin‑remodeling machinery,the recombinant of actin stimulates the fusion of lysosome and autophagosome through assembly of an F-actin network, to degrade the substrates (Li et al, 2018; Tabas and Ron 2011). An example, inhibition of HDAC6 weakens autophageal induced by bortezomib in HNSCC cells (Chang and Wang C, 2016), Tubacin, an inhibitor of HDAC6 which induces autophagy and blocks the fusion of autophagosom 103; (Yin and Li, 2018). Autophagy is classified into macro-autophagy, micro-autophagy, and Chaperone-mediated autophagy (CMA) according to the approach to transport proteins and whether bi-membrane is formed. CMA is activated constitutively and maximally under oxidative stress (Kaushik and Cuervo , 2006), providing a front defense against oxidative stress (Scherz-Shouval and Elazar, 2007) . Knockout or knockdown of HDAC6 induces irreversible hyperacetylation of HSP90, leading to lowering activity of CMA, and weakens the resistance to ROS of the cells (Shen et al., 2009) .
Some study suggests that HDAC6 inhibition increases autophagy. In acute renal injury model of rat, to autophagosome formed by components Atg7 and Beclin-1, shows remarkably elevation in injured kidneys. Treatment with Tubastatin A further increases the expression amount of Atg7 and Beclin-1, indicating the autophagy due to renal injury is increased by HDAC6 inhibition (Tang et al, 2018). In CAL27 cells, a HDAC6 inhibitor Ricolinostat treatment for 24 h, cytoplasmic autophagosome and lysosomes increase significantly in amount, suggesting that Ricolinostat induces autophagy in CAL27 cells(Hattori, 2021)]. Other study reveals that HDAC inhibition promotes autophagy through activation of transcription factor EB (TFEB) (Brijmohan , et al. 2018) . Further investigation reveals that after treatment with hydrogen peroxide the rat chondrocyte antioxidase and apoptosis induced by Tubastain A is inhibited by autophagy inhibitor 3-MA, demonstrating the HDAC6 mediated anti-oxidative that involves autophagy (Shen et al., 2021).
3.4 HDAC6 and Nrf2
Transcription factor Nrf2(nuclear factor red cell 2 related factor)is a transcription factor rich in leucine zipper, belonging to CNC transcription factor family, is a central regulator of cellular stress (Choi, et al, 2018),Nrf2 is expressed in various tissues and cells, to provide protection against cell injury, notably of oxidative stress and disequilibrium of oxidation and reduction (Wakabayashi et al, 2010) . Oxidative and pro-electron agents bind Keap1 (Kelch-like ECH-associated protein 1), gives rise to accumulation and translocation of Nrf2 in the nuclei (Zhang et al, 2004). Nrf2 contains a basic leucine zipper (bZIP) on its C-terminus,functioning as DNA binding domain. It dimerizes with Mafin the nuclei, to recognize and bind sequence GCTGAGTCA on anti-oxidation module ARE, and initiate transcription and upregulation of cell protective andante-oxidative enzymes, mediated by ARE, including cysteine S transferase (CST), catalase (CAT), thioredoxin(TRX), superoxidase (SOD), Heme oxygenase-1(HO-I), NAD(P)H , quinone oxidoreductase-1(NQO1) and other anti-oxidases (Han, et al. 2017). Their expression help to clear ROS, increase synthesis of cysteine, and reduce ketone chemicals, so as to protect cells from oxidative stress and maintain equilibrium of intracellular partial oxygen in the cells.
HDAC6 has been proved to inhibit transcription factor Nrf2. In LPS stimulated RAW264.7 cells,Nrf2 and the downstreamprotein HO-1 restores to normal level through inhibition of HDAC6 (Zhang et al., 2019a). Nrf2 modulates the level of HDAC6, and HDAC6 is persistently upregulated in cells genetic oxidative sensitive model mice with Nrf2 knockout (Lee, et al. 2010) .
Multiple investigations reveal that, HDAC6 modulate Nrf2 so as to ameliorate injury due to oxidative stress. With HDAC6 knockout, MCAO mice show remarkably elevated level of Nrf2 and HO-1 in both cytosol and nuclei, suggesting that HDAC6 interference not only promotes production of Nrf2, and accelerate its transport to nuclei from cytoplasma, meanwhile it also reduces production of the oxidative stress markers in the serum, like 3-NT、4-HNE、8-OHdG, to protect mice from injury of oxidative stress through activating of Nrf 2 pathway with HDAC6 inhibition (Li et al, 2019) . Other study suggests that the level of Nrf2 dependent genes during diabetes, like: HO-1, NQO1, glutamate-cysteine ligase regulatory subunit(GCLM) and glutamate-cysteine ligase(GCLC) is recovered by Tubastatin A, and the same with oxidative markers DHE, NT, 4-HNE, Trx1,suggesting that Tubastatin A reduces oxidative stress by restoring Nrf 2 dependent pathway in diabetic retina ( Abouhish et al., 2020) .
3.5 HDAC6 and NADPH oxidase
NADPH oxidase is the potential source of intracellular ROS; it is composed of membrane association subunit (gp91phox/Nox2、p22phox) and cytoplasmic subunit (p40phox, p47phox, p67pho, small GTPase Rac) (Lambeth , et al., 2007). On activation, the cytoplasmic subunit is transferred to the membrane to bind gp91phox/Nox2, then it catalyzes NADPH dependent oxygen to superoxide (Brandes et al., 2014, Youn et al., 2017) .
Available data suggests that HDAC6 modulates the activities of NAPDH oxidase to reduce oxidative stress induced injury. In LPS stimulated primary bovine mammary epithelial cells (bMECs),Inhibition of HDAC6 by Tubastatin A reduces the production of NADPH oxidase,meanwhile the generation of ROS (Wang et al., 2018). In RAW 264.7cells,overexpression of HDAC6 leads to elevation of both NAPDH oxidase and ROS,and combined with high dose of HDAC6 inhibitor of NAPDH oxidase reduces the production of ROS,directly indicates that ROS elevation is by HDAC6 is in part mediated by NAPDH oxidase. In addition, overexpression of HDAC6activates MAPK/NF-κB/AP-1signaling pathway,and inhibitors of ROS and NADPH oxidase blocks the activation of MAPK suggesting that overexpression of HDAC6 influences ROS-MAPK-NF-κB/AP-1pathway to modulate inflammation through upregulation of NADPH oxidase (Youn et al., 2016) . Further study reveals that knockout of HDAC6 and treatment with Tubastain A significantly inhibits HIV-1 Tat induced NADPH oxidase activity and elevated ROS elevation in CRT-MG human astroglial cells. Meanwhile,inhibitors of HADPH oxidase and ROS also reduces expression of HDAC6 in HIV-1 Tat stimulated asterogliocytes,suggesting of interactions between NADPH oxidase, ROS and HDAC6 (Youn, et al., 2017) .
3.6 HDAC6 and XBP1
XBP1s (X box-binding protein-1) is a leucine zipper transcription factor participating mammalian UP, playing an important role in anti-oxidative effect by HDAC6 inhibitor Tubastain A. The accumulation of misfolded proteins due to endoplasmic reticulum(ER) errors triggers ER stress (ERS) and causes unfolded protein response (UPR)(Schröder and Kaufman, 2005) . Production of ROS is often related to transcription activation induced by ERS. Research indicates that,Tubastatin A and tubacin treatment increase the level of XBP1s and their acetylation in a dose and time dependent manner, while the half-life of XBP1s prolongs about 30 min. after Tubastatin A treatment,and the expression of downstream genes significantly increases. The activities of XBP1s are elevated after concurrent challenge with H2O2and Tubastatin A, and inhibition on XBP1s with siRNA reduces the upregulation of anti-oxidative genes caused by Tubastatin A, directly indicating the involvement of anti-oxidative activity by HDAC6 (Zhang et al., 2014).
The processes that HDAC6 participates to ameliorate oxidative stress are depicted in Fig. 1.
3.7 Other oxidative stress pathways related to HDAC6
In addition to the molecules and pathways discussed above, there are some molecules which indirectly contribute to the HDAC6 mediated oxidative stress modulation. The role in the process remains to be validated.
3.7.1 Heat shock protein (HSP)
Cells produce HSP in response of stress (Stahnke et al., 2007). HSP are highly conserved proteins, serving as molecular chaperone and first line defense of stress. They are induced by heat shock factor 1 (HSF1). In unstimulated cells,HSF1 is present in inhibitory complex of HDAC6 and HSP90 (Leyk et al., 2017). During stress, HSF1 is dissociated from the complex, activated and migrates to nuclei and binds heat shock protein coding genes, to initiate their expression (Pernet et al., 2014).
HSP is inducible by oxidative stress (Gorman et al., 1999). Martindale and Holbrook, 2002). HSP25 and HSP70 play an important role on retina degeneration protection from oxidative stress (O'Reilly et al., 2010, Kim et al., 2015) ,HSP90 is a molecular chaperone protein playing a key role in defense of oxidative stress (Kim et al., 2015),to increase HSP90 synthesis determines a more potent defense against ROS induced injury; its degradation is determined by the acetylation status( Leyk et al., 2017).
HDAC6 is linked with activation of HSP gene,Treatment of 661W with Tubastatin A, the cells upregulateHSP70 and HSP25 through activating HSF1. Besides, the inhibition of HDAC6 causes a chaperone function of HSP90 through a super-acetylation (Leyk et al., 2017). In K-562 and HL-60 cells, HDAC6 inhibitor tubacin increases the level of HSP90 acetylation; the ROS inhibitor NAC reduces HDAC6, HSF1, HSP27, HSP70 and HSP90,suggesting that oxidative stress is an effective inducer of HDAC6 and HSPs ( Sarkar et al., 2014).
When co-incubating 661W cells with KRIBB11, an activating inhibitor of HSF1, HSP70 and HSP25 induction are inhibited under oxidative stress. The protection of Tubastain A against H2O2 induced cytotoxicity, however, is not weakened, suggesting that these HSPs do not exerting actions alone is not sufficient to prevent oxidation mediated injury, and contribution of other factors is required(Leyk et al., 2017).
3.7.2 NLRP3 inflammasome
NLRP3 is a member of Nod-like receptor family, with function of sensing pathogens and injury. It forms a cytoplasmic multi-protein complex with apoptosis related micro-particle protein ASC and caspase-1, to modulate the activation of IL-1β and IL-18 (Mangan et al., 2018). Most confirmed NLRP3 activators also stimulate production of ROS. Anti-oxidants are also proved to inhibit the activation of NLRP3, indicating that redox signals participate its activation (Martinon 2010). Relevant study also indicates that, the inactivation of NLRP3 inflammasome helps cells recover from oxidative stress, inflammation and apoptosis induced by high glucose (Lu et al., 2018, Li et al., 2019) . The treatment of HDAC6 inhibitor Cay downregulates,NLRP3and oxidative stress as well as inflammation indicators. The data suggests that, Cay may inhibit oxidative stress, inflammation, and apoptosis through modulation of inflammatory pathway of NLRP3 (Yang et al., 2002). The pharmacologic inhibition of HDAC6 by Tubastatin A inhibits the activation of NLRP3 production of ROS by 6-OHDA in SH-SY5Y, human neuroblastoma cell line or mice nigrostriatal system and weakens the neurotoxicity of dopamine energistic neuron (Yan et al., 2020).
3.2.3 NF-κB pathway
Nuclear factor-κB (NF-κB) is a transcription factor with multiple regulatory functions in the cells (Xia et al., 2014). Its activation is modulated by IκB kinase and phosphorylation and degradation of IκBα, tow components in NF-κB family (Natoli and Chiocca S, 2008). ROS produced by macrophage activates the NF-κB pathway, and triggers inflammationto upregulate pro-inflammatory factors, chemotactic factors, adhesion molecules and NADPH (Youn et al., 2016, Kaul and Forman , 1996) .
The promotion activity in inflammation and oxidative stress of HDAC6 involves NF-κB pathway. In macrophages, overexpression of HDAC6 activates the NF-κB signaling pathway and expression of pro-inflammatory cytokines (Youn et al., 2016) . In LPS-stimulated RAW264.7 cells,inhibition of HDAC6 restores MAPK and NFκB pathways activation (Zhang, et al., 2019a). Other study indicates that in high glucose treated cells, inhibitor of HDAC6 Cay treatment significantly reduces the expression of nulcear NF-κB p65 and p-IκB-α but increases cytoplasmic NF-κB p65 expression,with concurrent reduction of oxidative, inflammation and apoptosis indicators, suggesting that HDAC6 inhibitors blocks oxidative stress, inflammation and apoptosis induced by high glucose induced through modulation of the NF-κB pathway (Yang et al., 2002). In LPS stimulated primary cultured bovine mammary epithelial cells (bMECs), Inhibition of HDAC6 with Tubastatin A reduces the production of ROS, reduces the phosphorylation level NF-κB p65 and IκB in a dose dependent manner (Wang et al., 2018) . In acute renal injury model of mice,NF-κB signal is remarkably increased in the mice kidneys, with elevated Il – 6 and TNF-α expression. The pharmacologic inhibition of Tubastatin A to HDAC6 significantly reduces the abnormal phosphorylation and activation of NF-κB and expression of inflammatory factors. The data indicates that after renal injury, Tubastatin A, an inhibitor to HDAC6 reduces NF-κB phosphorylation, and expression of IL-6 and TNF-αto relieve renal inflammation (Shi et al., 2017).
3.7.4 MAPK-ERK pathway
MAPK-ERK signaling pathway is a complicated network; it is of essential role in physiologic and pathologic activities,involving a series of cellular response triggered by environmental and developmental signal,including cell survival, proliferation, differentiation, inflammation and apoptosis (Pearson et al., 2001) . In inflammation, MAPK and other transcription factors are strictly regulated in an oxidation-reduction dependent manner (Song and Brady S, 2015) .
It has been demonstrated that HDAC6 and the MAPK-ERK signaling pathway possesses a close relationship. ERK1/2 as the end effectors of ERK-MAPK pathway interacts with HDAC6 in vivo (Williams et al., 2013) . ERK1/2 phosphorylates HDAC6 at Serine 1035 (Williams et al., 2013) , and HDAC6 deacetylates p38 and then ERK1/2 phosphorylates it, in response of proteasomal inhibition ( Kastle et al., 2012) . In addition, JNK1 maintains the expression of HDAC6 ( Zhang et al., 2010).
A study indicates that overexpression of HDAC6 stimulates MAPK signaling pathway, and the effect is blocked by ROS inhibitor, suggesting that overexpression of HDAC6 modulates MAPK pathway through upregulation of ROS (Youn et al., 2016). Other study suggests that H2O2increases ERK1/2 and AKT in HUVECs cells, and concurrently reduces HDAC6. The subsequent treatment with Erk1/2 inhibitor U0126 causes increase of HDAC6 indicating that H2O2 reduces the amount of HDAC6 through stimulating ROS dependent on the Akt and ERK1/2signaling pathways ( Cai et al., 2018).
3.7.5 AKT pathway
AKT widely participates various processes in the cells, acting as an important physiologic modulating pathway. It has been demonstrated to be associated with oxidative stress and HDAC6. A study suggests that the total amount of AKT and phosphorylation increases in an mice renal injury model, and it is inhibited by Tubastatin A revealing that HDAC6 inhibition reduces phosphorylation of AKT (Tang et al., 2018) . It has been proved that GSK-3β phosphorylates Ser-22 of HDAC6 (Chen et al., 2010). When exposing to extract of tobacco smoke, in vitro cultured lung tissue and mice lung tissue show lower phosphorylation of Akt ser473 and GSK-3β ser9, and elevated level of phosphorylation in HDAC6 ser22; the changes are reversed by ROS inhibitor NAC. Meanwhile tobacco smoke reduces level of ac-tubulin, suggesting that inactivation of Akt and the subsequent GSK-3β activation are caused by oxidative stress; and that activated GSK-3β activatesHDAC6 through phosphorylation of Ser-22, reducing ac-tubulin and assembly of microtubule to injure lung endothelium ( Borgas et al., 2016) .
Actions of molecules discussed in the section 3.7 are summarized in Fig. 2.