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Objective To investigate the protective mechanism of Wenyang Fuyuan Prescription on nerve injury by improving brain iron metabolism in rats with cerebral ischemia-reperfusion injury(CIRI)based on ferroptosis.Methods A total of 72 SD rats were randomly divided into sham-operation group,CIRI model group,Wenyang Fuyuan Prescription group(18.0 g·kg-1,gavage),ferroptosis inducer group(100 mg·kg-1,intraperitoneal injection),Wenyang Fuyuan Prescription(18.0 g·kg-1,gavage)+ ferroptosis inducer group(intraperitoneal injection)and ferroptosis inhibitor group(5 mg·kg-1,intraperitoneal injection),12 rats in each group.All the procedures adopted in the sham group were the same as those in the model group.But nylon thread was inserted into the internal carotid artery at a depth of 9 mm and un-plugged middle cerebral artery.The rest of the groups were used to construct middle cerebral artery occlusion/reperfusion(MCAO/R)model by thread embolism method.Ferroptosis inducer(100 mg·kg-1)and ferroptosis inhibitor(5 mg·kg-1)were administered intraperitoneally to rats according to the grouping 24 hours before modeling.Wenyang Fuyuan Prescription(18.0 g·kg-1)was administered by gavage 2 hours after anesthesia and awakening.All intervention were given once daily for 7 consecutive days.The Longa scoring standard was used to evaluate the neurological deficit on 1,3,and 7 days after MCAO/R surgery,respectively.At the end of the treatment period,brain tissues were taken to observe the morphological changes of rat neurons in each group by hematoxylin eosin staining(HE).The ultrastructural changes of neuron mitochondria in each group were observed by transmission electron microscope.The biochemical kit was used to detect the content of iron ions(Fe2+)and reduced glutathione(GSH)in brain tissue.The protein and mRNA expressions of transferrin receptor 1(TFR1),iron regulatory protein 1(IRP1)and ferroportin(FPN)were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot.Results① Compared with sham group,the neurological deficit scores of rats in model group increased at each time point(P＜0.01).HE staining showed neurons were sparse and disordered,the nuclei underwent pyknosis,and vacuoles appeared at the edges.Under electron microscopy,it was observed that the number of neuronal mitochondria decreased,the density of mitochondrial membranes increased,massive numbers of mitochondrial membranes ruptured and dissolved,and mitochondrial cristae disappeared.The content of Fe2+,both mRNA and protein expressions of TFR1 were significantly increased(P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN were significantly decreased(P＜0.05,P＜0.01).② Compared with the model group,the neurological deficit scores of rats in the Wenyang Fuyuan Prescription group decreased at various time points(P＜0.05).The number of neurons increased,their arrangement was relatively neat,the morphology of the nucleus is complete and clear,the mitochondrial structure of neurons was relatively complete,the mitochondrial membrane was relatively intact,and the mitochondrial cristae were clear.The content of Fe2+,both mRNA and protein expressions of TFR1 were decreased(P＜0.05,P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN increased(P＜0.05,P＜0.01).③ Compared with the Wenyang Fuyuan Prescription group,the neurological deficit scores of rats in ferroptosis inducer group and the Wenyang Fuyuan Prescription + ferroptosis inducer group increased at all time points(P＜0.05).Distribution of neurons was in disorder,the nucleus shrinked,and vacuoles appeared at the edges.The density of mitochondrial membranes increased,some ruptured and dissolved mitochondrial membranes were found.The number of mitochondria decreased and mitochondrial cristae disappeared.The content of Fe2+,both TFR1 mRNA and protein expression increased(P＜0.05,P＜0.01),while the content of GSH,as well as expressions of mRNA and protein for IRP1 and FPN decreased(P＜0.05,P＜0.01).However,there was no statistically significant difference in all observed indicators between the ferroptosis inhibitor group and the Wenyang Fuyuan Prescription group(P＞0.05).Conclusion Wenyang Fuyuan Prescription can improve the neurological function and pathological damage of CIRI rats.Its mechanism may be related to regulating the expression of IRP1 protein,improving the brain iron metabolism pathway,and inhibiting ferroptosis.
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@#Objective To investigate the protective mechanism of Rho protein kinase 2 (ROCK2) inhibitor constructed based on adeno-associated virus vector on vascular dementia (VaD) rat hippocampal neurons.Methods Forty SD rats were randomly divided into sham operation group,model group,ROCK2 interference group (shROCK2),negative control group.The VaD model was prepared by permanent ligation of bilateral common carotid arteries.AAV9-ROCK2-shRNA was injected into the hippocampus of the rat by stereotaxic technique,the negative control group was injected with the same amount of empty vector adeno-associated virus.After 4 weeks,morris water maze was used to test the learning and memory abilities of rats.RT-PCR and Western blot were used to detect the mRNA and protein expression levels of RhoA,ROCK2,MLC,MLCP.Results Four weeks after operation.The mRNA and protein expressions of the above 4 indicators in hippocampus increased (P<0.01),the synaptic vesicles disappeared,and the structure of hippocampus was chaotic in the model group.Compared with the model and control group,the learning and memory ability of rats in ROCK2 interference group was significantly improved,the mRNA and protein of the above 4 indicators were significantly decreased (P<0.05),and the pre-synaptic vesicles were normal and the organelles were more complete.Conclusion Rho protein kinase 2 inhibitors can reduce the expression of downstream related proteins by inhibiting RhoA/ROCK2 signaling pathway,then promote neuronal axon regeneration and alleviate cognitive dysfunction of VaD rats.
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This study was aimed to explore the function of c-Jun N-terminal kinase (JNK) signaling pathway in the induction of brain ischemic tolerance, and observe the function of Shu-Xue Tong-Mai (SXTM) capsule pretreatment. Ischemic preconditioning was performed for 3 min on rats to induce cerebral ischemic tolerance. Rat model of cere-bral ischemia reperfusion (the ischemia pretreatment group, I/R group) was established 24 h later. Western blot was used to detect the protein expression of JNK and phosphorylation of c-Jun N-terminal kinase (P-JNK), comparing to the expression with the sham operation group, I/R group and SXTM capsule group. Tunel method was applied to de-tect the apoptosis of neurons. Relationship between expression of JNK, P-JNK and apoptosis of neurons was also studied. The results showed that compared with the model group, expressions of P-JNK in ischemia preconditioning group and SXTM group were declined significantly (P < 0.05); and the apoptosis of neurons quantity was also de-clined (P< 0.05). It was concluded that ischemia preconditioning can decrease the apoptosis of neurons in cerebral ischemia reperfusion, and improve neurologic function. Its mechanism related to the inhibition of JNK signaling path-way. SXTM capsule pretreatment can protect the cerebral by inhibiting the JNK signaling pathway.
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ObjectiveTo investigate the effect of focal ischemic preconditioning (IPC) on the expression of protein kinase-like endoplasmic reticulum kinase ( PERK ) and glucose-regulated protein 78 (GRP78) mRNA and protein after focal cerebral ischemia/reperfusion (I/R) in rats.MethodsAll 120 male SD rats were randomly divided into three groups: sham-operation group,middle cerebral artery occlusion (MCAO) group and brain ischemia preconditioning (BIP) group.Each group was further divided into 4 subgroups according to 12 h,1,2 and 3 d after I/R.The IPC models were made in order to measure the expression of PERK,GRP78 mRNA and protein by in situ hybridization and Western blot,and the apoptosis rate of neuron by flow cytometry. Results ①The expression of PERK mRNA increased and reached the peak at 12 h,then decreased continuously after 1 d.BIP could decrease its expression.The expression of PERK protein increased at 12 h and reached the peak at 24 h,then decreased continuously after 2 d.BIP could decrease its expression.②The expression both of GRP78 mRNA and its protein all increased and reached the peak at 12 h,then decreased continuously.BIP could increase their expression (mRNA:12 h: 136.70±9.53,F=32.265; 24 h:147.54 ±9.97,F=54.920; 2 d:158.16 ±9.44,F=45.374; 3d: 165.85±10.26,F=16.493,P＜0.05; protein:12 h: 1.319±0.116,F=5.619,P＜0.05; 24 h: 1.226±0.108,F=33.742,P＜0.01; 2 d:1.183 ±0.112,F =46.556,P ＜0.01; 3 d:1.115± 0.098,F =11.730,P＜0.05).③The rate of apoptosis neuron of rats in MCAO increased markedly at 12 h after reperfusion,and reached the peak at 1 d,then decreased continuously.BIP could decrease the rate of apoptosis neuron. Conclusion BIP can protect neurons through inhibiting the expression of PERK and inducing the expression of GRP78 after endoplasmic reticulum stress in rats.