Design and synthesis of sulfonamide phenothiazine derivatives as novel ferroptosis inhibitors and their therapeutic effects in spinal cord injury.

Ferroptosis is a novel style of cell death, and studies have shown that ferroptosis is strongly associated with spinal cord injury (SCI). A large number of ferroptosis inhibitors have been reported, but so far no ferroptosis inhibitor has been used clinically. Therefore there is an urgent need to discover a better inhibitor of ferroptosis. In this study, 24 novel sulfonamide phenothiazine ferroptosis inhibitors were designed and synthesized, followed by structure-activity relationship studies on these compounds. Among them, compound 23b exhibited the best activity in Erastin-induced PC12 cells (EC50 = 0.001 µM) and demonstrated a low hERG inhibition activity (IC50 > 30 µM). Additionally, compound 23b was identified as a ROS scavenger and showed promising therapeutic effects in an SD rat model of SCI. Importantly, 23b did not display significant toxicity in both in vivo and in vitro experiments and show good pharmacokinetic properties. These findings suggest that compound 23b, a novel ferroptosis inhibitor, holds potential as a therapeutic agent for spinal cord injury and warrants further investigation.

Kapß2 Inhibits Perioperative Neurocognitive Disorders in Rats with Mild Cognitive Impairment by Reversing the Nuclear-Cytoplasmic Mislocalization of hnRNPA2/B1.

Harmful stimuli trigger mutations lead to uncontrolled accumulation of hnRNPA2/B1 in the cytoplasm, exacerbating neuronal damage. Kapß2 mediates the bidirectional transport of most substances between the cytoplasm and the nucleus. Kapß2 guides hnRNPA2/B1 back into the nucleus and restores its function, alleviating related protein toxicity. Here, we aim to explore the involvement of Kapß2 in neurodegeneration in rats with MCI following sevoflurane anesthesia and surgery. Firstly, novel object recognition test and Barnes maze were conducted to assess behavioral performances, and we found Kapß2 positively regulated the recovery of memory and cognitive function. In vivo electrophysiological experiments revealed that the hippocampal theta rhythm energy distribution was disrupted, coherence was reduced, and long-term potentiation was attenuated in MCI rats. LTP was greatly improved with positive modulation of Kapß2. Next, functional MRI and BOLD imaging will be employed to examine the AFLL and FC values of dynamic connectivity between the cortex and hippocampus of the brain. The findings show that regulating Kapß2 in the hippocampus region enhances functional activity and connections between brain regions in MCI rats. WB results showed that increasing Kapß2 expression improved the expression and recovery of cognitive-related proteins in the hippocampus of MCI rats. Finally, WB and immunofluorescence were used to examine the changes in hnRNPA2/B1 expression in the nucleus and cytoplasm after overexpression of Kapß2, and it was found that nucleocytoplasmic mis location was alleviated. Overall, these data show that Kapß2 reverses the nucleoplasmic misalignment of hnRNPA2/B1, which slows neurodegeneration towards dementia in MCI after sevoflurane anesthesia and surgery. Our findings may lead to new approaches for perioperative neuroprotection of MCI patients.

Gastrodin Prevents Neuronal Apoptosis and Improves Neurological Deficits in Traumatic Brain Injury Rats through PKA/CREB/Bcl2 Axis.

BACKGROUND: Gastrodin (Gas) exhibits anti-inflammatory properties against diseases associated with the central nervous system (CNS). This study aimed to investigate the potential neuroprotective role of Gas in traumatic brain injury (TBI). METHODS: A rat TBI model was established in male adult Sprague-Dawley (SD) rats by controlled cortical impingement (CCI), and lipopolysaccharide (LPS) was applied to induce the activation of BV2 microglia and HT22 hippocampal neurons. Neurological deficits, motor function and brain water content were evaluated in TBI rats. TUNEL and Nissl's staining were applied to measure neuronal degeneration and apoptosis. Microglial activation, the mRNA and protein profiles of pro-inflammatory cytokines were tested by immunohistochemistry (IHC), quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Gas significantly reduced neurological deficits, cerebral edema, and neuronal apoptosis and improved motor function in TBI mice. In addition, Gas inactivated microglia and blocked the production of pro-inflammatory cytokines on the damaged side of the TBI rat brain. In vitro, Gas attenuated BV2 microglia inflammation and reduced HT22 hippocampal neuronal apoptosis. On the other hand, Gas activated the PKA/CREB/BDNF pathway both in vivo and in vitro. CONCLUSIONS: Gas blocks microglial activation-mediated inflammation through the PKA/CREB/BDNF pathway, thereby improving neurobehavioral function after TBI, which provides a potential therapeutic benefit for treating TBI.

Effect of different therapeutic time windows of mild hypothermia on neurological protection from ventricular fibrillation in rabbit models / 中华急诊医学杂志

Objective To investigate the effects of mild hypothermia on post-resuscitation neurological outcome after ventricular fibrillation (VF) in rabbits.Methods Forty-five adult New Zealand rabbits were induced VF by direct current of electricity.The rabbits were randomly(random number) divided into following groups:normothermic resuscitation group (NR),mild hypothermia prearrest group (HP),mild hypothermia resuscitation 30 min group (HRe30),mild hypothermia resuscitation 90 min group (HRe90),normothermic sham group (NS),and hypothermia sham group (HS).The rabbits of NR group were observed for 600 min in room temperature after restoration of spontaneous circulation (ROSC).The mild hypothermia was induced by surface cooling,and maintained for 600 min after the aimed low temperature reached.The arterial blood samples were collected for determining neuron-specific enolase (NSE) and thioredoxin (Trx) and the mean arterial pressure (MAP),left ventricular end-diastolic pressure (LVEDP) and left ventricular pressure raise and fall rate (±dp/dtmax) were observed at 15 min before CA,and 30 min,60 min,120 min,360 min and 600 min after ROSC.After the animals were sacrificed at 600 min after ROSC,the whole brain of animals was harvested and observed under light microscope to calculate the apoptotic index of the hippocampal CA1 neurons by using TUNEL method.One-way ANOVA was used to determine the statistical significance between two groups,a two-tailed value of P＜0.05 was considered statistically significant.Results (1) Hemodynamically compared with normal temperature groups,HR was lower in hypothermia groups.Compared with NR,HRe30,and HRe90 group,LVEDP was higher in HP group at 30 min after ROSC(3.4±0.8 vs.4.6±1.0,4.1±0.5,4.3±0.2,F=9.85,P=0.019).In Hp group,the level of +dp/dtmax was higher than that in NR,HRe30 and HRe90 groups at 30 min and 120 min after ROSC.In HP group,the level of-dp/dtmax was higher than that of NR group at 30 min,60 min,120 min,360 min and 600 min after ROSC.(2) Serologically compared with HP,HRe30 and HRe90 group,NSE levels were higher in NR group at 60 min,120 min and 360 min after ROSC.Compared with NR,HRe30,and HRe90 group,Trx levels in NR group were lower at 60 min,120 min,360 min and 600 min after ROSC.Compared with HP group,Trx levels in HRe30 and HRe90 groups were higher at 60 min,120 min,360 min and 600 min after ROSC.(3) Pathologically compared with NR group,histopathological changes in hippocampus CA1 area were milder found in HP,HRe30 and HRe90 groups.AI (％) was lower in HP,HRe30 and HRe90 groups than that in NR group[(62.25±10.43)％ vs.(20.61±5.02)％,(25.08±3.92)％,(30.33±7.15)％,P=0.001].Concusions This study shows that hypothermia should be initiated as soon as possible,and especially early intra-arrest cooling appears to be significantly better than post-ROSC cooling and normothermia.

Repair of Neurological Function in Response to FK506 Through CaN/NFATc1 Pathway Following Traumatic Brain Injury in Rats.

Tacrolimus (FK506), an immunophilin ligand, has been widely shown to be neuroprotective in a posttraumatic period. The nuclear factor of activated T cells (NFATc1) pathway plays an important role in regenerating neurological function following traumatic brain injury (TBI), but the precise mechanism underlying FK506-induced repair of neurological functions remains unclear. In the present study, a total of 210 SD rats were enrolled and randomly divided into sham group, TBI group and FK506 group. The rats in the TBI and FK506 groups were inflicted with moderate TBI left lateral fluid percussion impact. A modified neurological severity score (mNSS) system was used to evaluate the severity of effects on nerve function. mNSS levels were significantly lower in the FK506 group than in the TBI group. The zaccumulation of cerebral water content was lower, cerebral Aquaporin 4 (AQP4) mRNA level was lower, the number of growth-associated protein-43 (GAP-43)-positive cells was higher, and the distribution of vesicles containing excitatory neurotransmitters was altered in the injured cortex in the FK506 group. Moreover, the cortical mRNA and serum protein expression levels of interleukin-2 (IL-2) and interferon-γ (IFN-γ) were decreased in FK506 group, especially at 6 h and at 1 day after TBI. At days 1-28 after TBI, the expression of cleaved-caspase 3, which indicates apoptosis, was lower in the FK506 group than in the TBI group. Mechanistically, FK506 significantly down-regulated the mRNA and protein levels of calcium-regulated phosphatase (calcineurin, CaN) and inhibited the activation of NFATc1. These results demonstrate that FK506 relieved inflammatory responses by regulating the NFATc1 signaling pathway and promoting the synaptic reconstruction of neurons and glial cells by regulating cell apoptosis, thereby facilitated improvements in neurological function.

The effect and mechanism of rt-PA combined with high pressure oxygen on cerebral ischemia-reperfusion inj ury in rats / 西安交通大学学报(医学版)

Objective To evaluate the effect and mechanism of rt-PA combined with high pressure oxygen (HPO)on cerebral ischemia-reperfusion injury in rats.Methods The model of cerebral ischemia-reperfusion injury was constructed by using middle cerebral artery occlusion.The neurological function score;brain index,water content and infarction volume;SOD;LDH;NOS;MDA;LD;NO and NOS were measured.The protein and mRNA expressions of iNOS,BDNF,p75NTR and TrkB were also detected by RT-PCR and Western blot to evaluate and compare the protective effect of rt-PA combined HPO therapy. Results rt-PA combined HPO could significantly decrease the neurological function score;brain index,water content,and infarction volume;SOD;NOS;MDA;LD;NO and NOS but increase LDH content and the weight of rats,compared with rt-PA.In addition,rt-PA combined HPO could increase BNDF and TrKB expressions and downregulate the expressions of iNOS and p75NTR,compared with rt-PA (P<0.05).Conclusion The rt-PA combined HPO therapy has a greater protective effect than rt-PA therapy and its mechanism might be related to having antioxidant effects, increasing the expressions of BDNF and TrKB,and decreasing the expressions of iNOS and p75NTR.

The effect of insulin on the expressions of Bcl-2, Bax mRNA and hippocampus neuronal apoptosis in rats after cardiopulmonary resuscitation / 中华急诊医学杂志

Objective To explore the effects of intraventricular administration of insulin on the expressions of Bcl-2,Bax mRNA and neuronal hippocampus apoptosis in rats after cardiopulmonary resuscitation (CPR).Methods This experiment was implemented in the animal Laboratory center of Xuanwu Hospital of Capital Medical University.Thirty male SD rats were randomly (random number)divided into three groups:control group (n=6),CPR group (n=12),insulin treated group ( n =12).CPR was performed at 6 minutes after ventricular fibrillation induced by transesophageal overdrive pacing.Resuscitation procedures lasted until restoration of spontaneous circulation (ROSC).ROSC was defined as the recovery of the supraventricular heart rates and the increase of mean arterial pressure (MAP) ＞ 60mmHg for more than 10 minutes.Ten minutes after ROSC in rats,12.5 μL ( 1 U) regular insulin was injected into the left ventricle in the insulin group,and 12.5 μL isotonic saline was injected the control and CPR groups at least 10 minutes.Real-time PCR was used to observe the expressions of Bcl-2,Bax mRNA in hippocampus CAI after reperfusion 24 h and 72 h.TUNEL staining was used to observe the neuronal apoptosis in all groups after reperfusion 7 days.Blood glucose was monitored in rats before and after CPR.Results ① The Bcl-2mRNA in insulin groups were significantly higher than those in the CPR group after 24 h and 72 h (P ＜0.01 ).The expression of Bcl-2 mRNA in 24 h insulin group were significantly higher than those in 72 h insulin group ( P ＜ 0.01 ) ; There were no significantly different in the Bax mRNA between insulin groups and the CPR and the control group after 24 h and 72 h ( P ＞ 0.05 ) ; ②After CPR 7 d,the apoptotic neurons of hippocampal CA1 area in the CPR group ( 124.75 ± 17.35 ) were significantly higher than those in the control group (5.12 ± 3.26) ( P ＜ 0.01 ) and the insulin group (92.79 ± 7.35 )(P ＜0.01 ); the apoptotic neurons in the insulin group were higher than those in the control group (P ＜0.0l ),and the differences were statistically significant.③There were no significant difference in venous blood glucose in the CPR and insulin groups (P ＞ 0.05).Conclusions Insulin may regulate Bcl-2mRNA expression in hippocampus,inhibit neuronal apoptosis and protect neurons after CPR in rats.