Neuroprotective Effects Of HDAC6 Inhibiting On Intracerebral Hemorrhage Through Regulating Autophagy And Nrf2 Pathway

ObjectiveTo assess neurological protecting effects of inhibiting HDAC6 synthetically.To measure expression of p62 and LC3-II before and after HDAC6 inhibitor administration,so as to invest HDAC6’s regulating mechanisms on autophagy.To invest HDAC6’s regulating mechanisms on Nrf2-HO-1 pathway through detecting expression of Nrf2 and HO-1under normal and HDAC6 inhibiting state.Methods1.Time points and dosages of Tub A administration;Assessment of protecting effectsHDAC6 was detected by Western Blot.Modified Neurologic Severity Score(m NSS)was utilized to assess the severity of rat neurological function damage.Measurement of Hb absorbance by ELISA,H-E staning and Tunel staning were used to assess protecting effects of Tub A.2.Neuron ICH model and its neuroprotective effects of HDAC6 inhibiting in vitroLC3II and p62 were detected by Western Blot.Western Blot: Total protein was extracted,which concentration detected by BCA kit.Protein was denaturized,electrophoretively analyzed,trans-membraned,immunoreacted and visualization.3.Regulating function on autophagy of HDAC6 inhibition in vivo and in vitroGrouping: Control: cells without special handling,Hemin+si-NC,Hemin+HDAC6-si RNA.The relative content of p62 was detected by Western Blot.Fixed sample was dehydrated,lucidificated and smoothed.The paster was soaked,cleaned,added into attenuated antibody,incubated and observed under a microscope.4.Influence of HDAC6 inhibiting on expression of Nrf2 and HO-1 in vivo and in vitroGrouping: Animals: 1)Sham: Rats were sham operated;2)ICH+vehicle;3)ICH+Tub A.Cells: Control: cells without special handling,Hemin+si-NC,Hemin+HDAC6-si RNA.ICH modeling in vivo and in vitro,perfusion,Western Blot,cell culture,si RNA interfering and statistic analysis are stated in text.Results1.Time points and dosages of Tub A administration;Assessment of protecting effectsAfter inhibitor administration,rat fatality declined from 25% in group ICH+3d to 14.6% in group ICH+vehicle.m NSS declined from 11.0in group ICH+3d to 6.8 in group ICH+vehicle.Comparing to group ICH+vehicle,the absorption rate of hemoglobin in group ICH+Tub A increases,(P<0.05).Comparing to group ICH+vehicle,the destruction of brain tissue in group ICH+Tub A decreases significantly,cells align neater,inflammatory cell infiltrate lesser,edema decreases significantly,peri-lesion tissue recovers.Comparing to group Sham,the percentage of TUNEL positive cells in group ICH+vehicle increases significantly,(P <0.05).Comparing to group ICH+vehicle,the percentage of TUNEL positive cells in group ICH+Tub A decreases,(P < 0.05).2.Neuron ICH model and its neuroprotective effects of HDAC6 inhibiting in vivo and in vitroComparing to group Control,cell apoptosis rate in group Hemin+si-NC increases significantly.(P<0.05).Comparing to group Hemin+si-NC,cell apoptosis rate in group HDAC6-si RNA decreases,(P<0.05).3.Regulating function on autophagy of HDAC6 inhibition in vivo and in vitroComparing to group Sham,the expression of p62 in group ICH+vehicle decreases significantly,the ratio of LC3II/I increases,(P<0.05).Comparing to group ICH+vehicle,the expression of p62 in group ICH+Tub A increases,the ratio of LC3II/I decreases significantly,(P<0.05).Comparing to group Sham,the expression of HDAC6 in group ICH+vehicle increases significantly,(P<0.05).Comparing to group ICH+vehicle,the expression of HDAC6 in group ICH+Tub A decreases,(P<0.05).4.Influence of HDAC6 inhibiting on expression of Nrf2 and HO-1 in vivo and in vitroComparing to group Control,the expression of Nrf2 and HO-1 in group Hemin+si-NC increases,(P<0.05),indicating activated Nrf2-HO-1protective pathways.Comparing to group Hemin+si-NC,the expression of Nrf2 and HO-1 in group HDAC6-si RNA increases further,(P<0.05),indicating strengthened Nrf2-HO-1 protective pathways.Conclusion1.HDAC6 inhibiting presents significant effects on improving rat survival rate,decreasing m NSS,increasing absorption rate of hemoglobin,attenuating peri-hematoma pathological damage and reducing apoptosis rate of neurons.Appropriate dosage of HDAC6 inhibiting administration could significantly alter the expression of autophagy biomarker p62 and LC3 II,indicating its regulation of autophagic flux.2.HDAC6 inhibiting could promote the expression of Nrf2 and HO-1,thus strengthens Nrf2-HO-1 pathway’s antioxidant and neuroprotective effects.