The Effect And Mechanism Of HDAC6 Inhibition On Neural Injury After Intracerebral Hemorrhage

Background and objective:Despite recent progress in the treatment of intracerebral hemorrhage(ICH),the clinical outcomes remain unsatisfactory.Cerebral edema and neuronal death are the main factors leading to poor long-term prognosis after ICH induction.Histone deacetylase 6(HDAC6)is a characteristic member of the class Ⅱb subgroup of histone deacetylases.Recent studies have found that HDAC6 inhibition by the selective pharmacological inhibitors or specific gene interference resulted in neuroprotection in several types of central nervous system disorder models.However,its role in ICH remains unclear.Therefore,this work was designed to explore the effect of HDAC6 inhibition and the potential mechanism on nerve damage in ICH models.Methods:1.Construction of ICH model in vivo and in vitro:(1)The ICH SD rats were performed by injecting collagenase type Ⅳ through stereotaxic intrastriatal implantation;(2)The HBMECs were treated with hemin to induce an in vitro ICH model;(3)The SH-SY5 Y cells were treated with hemin to induce an in vitro ICH model.2.Experimental grouping:(1)The SD rats were stochastically allocated into four groups: sham,ICH+ vehicle,ICH + TubA 25mg/kg,and ICH + TubA 40mg/kg;(2)The HBMECs were divided into five groups: control,Hemin + vehicle,Hemin + TubA(3μM),Hemin + NC-siRNA and Hemin + HDAC6 siRNA;(3)The SH-SY5 Y cells were sorted into five groups: control,Hemin +vehicle,Hemin + TubA(3μM),Hemin + NC-siRNA and Hemin +HDAC6 siRNA.3.Experimental contents:(1)The protective effect of HDAC6 inhibition on in vitro and in vivo models of ICH:In vivo experiment: The protein levels and intracellular distribution of HDAC6 and its substrate acetylated α-tubulin in perihematomal tissues over time were observed.We assessed the neurological scores,histopathologic changes of brains and brain water content in each group;In vitro experiment:(1)The protein levels of HDAC6 and its substrate acetylated α-tubulin in Hemin-induced SH-SY5 Y cells at 24 and 48 h.The cell density of SH-SY5 Y cells in each group was observed under light microscopy,and the viability of SH-SY5 Y cells in each group was detected by CCK8.(2)The protein levels of HDAC6 and its substrate acetylated α-tubulin in Hemin-induced HBMECs at 24 and 48 h.The cell density of HBMECs in each group was observed under light microscopy,and the viability of HBMECs in each group was detected by CCK8.(2)The potential mechanism of HDAC6 inhibition in protecting ICH(Ⅰ) — — regulating the acetylation level of α-tubulin to stabilize the blood-brain barrier(BBB)structure:In vivo experiment: The BBB permeability and ultrastructural changes of tight junctions(TJs)were observed in each group.And we tested the expressions of HDAC6,acetylated α-tubulin,tight junction proteins(occludin,ZO-1)and filament actin(F-actin)in each group.In vitro experiment: The HBMECs permeability and the ultrastructure of cell-cell tight junctions were observed in each group.And we tested the expressions of HDAC6,acetylated α-tubulin,tight junction proteins(occludin,ZO-1)and filament actin(F-actin)in each group.(3)The potential mechanism of HDAC6 inhibition in protecting ICH(Ⅱ) ——regulating the bcl-2/Bax/caspase3 pathway to improve neuronal apoptosis:In vivo experiment: The neuronal apoptosis rate and the proteins(Bax,bcl-2 and cleaved caspase-3)related to the signal transduction pathways for neuron apoptosis in perihematomal tissues of rats in each group.In vitro experiment: The neuronal apoptosis rate and the proteins(Bax,bcl-2 and cleaved caspase-3)related to the signal transduction pathways for neuron apoptosis of SH-SY5 Y cells in each group.Results:(1)In the rat model: we found a significant increase in HDAC6 during the early stages of ICH,which dramatically increased 1 d after ICH,and peaked at 3 days.The acetylated α-tubulin significantly decreased in correlation with the expression of HDAC6.HDAC6 was primarily distributed in the cytoplasm of neurons.We barely found HDAC6 co-localization with astrocytes and microglia.Medium and high doses(25,40 mg/kg)of TubA both reduced neurologic deficit,histological impairments,ipsilateral brain edema in vivo(P < 0.05).In the SH-SY5 Y cell model or HBMEC cell model: the expression of HDAC6 significantly increased and acetylated α-tubulin significantly decreased after 24 h treatment with hemin(P < 0.05).Compared to the Hemin group,the TubA treatment group or HDAC6 siRNA group showed higher cell densities under light microscopy,and the CCK8 results also indicated a higher viability of SH-SY5 Y cells and HBMECs in these two groups(P < 0.05).(2)In the rat model: compared with the ICH group,TubA groups significantly reduced the BBB permeability,the basal membrane and tight junctions in TubA groups were more integrate and regular under ultrastructural observation.In High-dose TubA group,the expression of HDAC6 was significantly reduced and the level of acetylated α-tubulin was significantly upregulated.And High-dose TubA rescued the degradation of tight junction proteins(ZO-1 and occludin,P<0.05).Moreover,treatment with 25mg/kg and 40 mg/kg dosage of TubA both inhibited actin stress fiber formation.In HBMECs model: compared with the ICH group,HDAC6 inhibition by TubA or HDAC6 siRNA reduced endothelial permeability of HBMECs after hemin induction(P < 0.05).The basal membranes in HDAC6 siRNA group or TubA group were more continuous and smoother.Moreover,TJs was longer and appeared as higher electronic density between adjacent cells.The expression of HDAC6 was significantly reduced and the level of acetylated α-tubulin was significantly upregulated in HDAC6 inhibition groups.Additionally,the inhibition of HDAC6 siginificantly attenuated the degradation of tight junction proteins(ZO-1 and occludin)and inhibited actin stress fiber formation(P < 0.05).(3)In the rat model: compared to the ICH group,the apoptosis rate of neurons in the two doses of TubA treatment groups were significantly reduced(P < 0.05).The high-dose TubA group showed a significant decrease in Bax and cleaved caspase-3,a significant increase in bcl-2(P< 0.05).In SH-SY5 Y cells model: HDAC6 inhibition by TubA or HDAC6 siRNA inhibited apoptosis of SH-SY5 Y cells after hemin induction(P <0.05).Moreover,TubA or HDAC6 siRNA group both significantly increased the acetylated α-tubulin and Bcl-2 while reducing the expression of HDAC6,Bax and cleaved caspase-3 after hemin induction(P < 0.05).Conclusion:HDAC6 inhibition has two protective effects in ICH:(1)It can regulate the expression of tight junction proteins(ZO-1 and occludin),stabilize the cytoskeleton F-actin and reduce the formation of stress fibers via upregulation of acetylated α-tubulin,thus playing a protective role of blood-brain barrier after ICH.(2)It can regulate the expression of apoptosis related proteins(bcl-2/Bax/caspase3),inhibiting neuronal apoptosis,thus exerting neuroprotective effects after ICH.