| As of 2013,according to relevant data,the prevalence of epilepsy in China is about 7.2 ‰,according to which it is estimated that there are about 9 million epilepsy patients in China,and about 400,000 new epilepsy patients are newly added each year(diagnosis and treatment of epilepsy).Epilepsy is a paroxysmal disease.Recurrent seizures cause severe neurological dysfunction,and epileptic seizures can cause death.Antiepileptic drugs are still the most basic treatment for epilepsy.In recent decades,the emergence of a variety of new anti-epileptic drugs has significantly improved the tolerance of drugs,and about 30% of patients with epilepsy can not be controlled by drug treatment.It puts a huge burden on patients and their families.Therefore,the study of epilepsy has considerable practical significance.The pathogenesis of epilepsy is complex,and the state of epilepticus causing neuronal death and its molecular mechanisms are still unclear.In recent years,great progress has been made in the study of temporal lobe epilepsy.An important pathological change in temporal lobe epilepsy is hippocampal sclerosis,which is characterized by neuronal loss and gliosis.At present,there are mainly the following viewpoints: abnormal neuronal potential,abnormal ion channels,imbalance of neurotransmitters in the central nervous system,abnormal immune function,apoptosis,and genetic abnormalities.It is generally believed that status epilepticus can induce excessive release of glutamate,activate glutamate N-methyl-D-aspartate receptor,trigger calcium ions to flow into neurons,activate nitric oxide synthase,and generate a lot of freedom.Activation of a variety of proteins associated with cell death ultimately lead to programmed cell death.Many studies on epilepsy at home and abroad have shown that seizures have the characteristics of staged progression,under the influence of various epileptic factors(cranial trauma,intracranial infection,intracranial parasitic infection,neurodegenerative diseases,etc.)In the early stage of seizures,reversible damage occurs in neurons,and the damaged cells can be rescued by treatment.In the middle and late stages of seizures,the damage caused by epilepsy to neurons will progress to irreversible damage.After applying antiepileptic drugs,merely a small number of neurons can be protected from necrosis.Therefore,the application of drug therapy in the early stages of seizures has become a new direction for the prevention and treatment of epilepsy.Baicalin is one of the main flavonoids in the traditional Chinese medicine Astragalus(Astragalus)(Molecular formula: C21H18O11)Baicalin has many different pharmacological effects,such as antioxidants,photoprotective agents,neuroprotective agents,antibacterial agents,anti-liver Toxicity and anti-cancer effects.Whether baicalin can protect hippocampal neurons by regulating autophagy in seizures has become a new exploration point.Our previous studies have shown that baicalin plays a neuroprotective role in seizures,and the specific mechanism has not yet been elucidated.The maintenance of cell homeostasis depends on the balance of biosynthesis and metabolism of macromolecular substances in the cell.This breaking of balance often leads to the self-regulation of cells to adapt to the environment.Autophagy function in the study of nervous system diseases has made a lot of breakthrough research,autophagy and neurodegenerative diseases,ischemic brain damage,central nervous system infection,immune abnormalities and epilepsy and other neurological diseases closely related.The word autophagy is derived from the combination of Greek,"auto"(self)and "phagy"(phagocytosis),the process of self-digestion of cells.So far,whether autophagy is involved in oxidative stress induced by seizures and what role it plays in neuronal death;the relationship between the protective mechanism of baicalin on seizures and autophagy has not been reported.In this study,a rat model of epilepsy induced by LiCl-PILO(pilocarpine,PILO)was established to investigate the pathological features of autophagy after hippocampal neuronal injury after seizure: the traditional Chinese medicine baicalin and autophagy inhibitor 3-methylindole were used respectively.(3-MA)intervened to detect changes in proteins and signaling molecules associated with autophagy,and to explore the role of autophagy in hippocampal injury induced by epilepsy in rats and its molecular mechanism.This study is divided into three parts.Part Ⅰ Observation of autophagy and neuron injuries in rat hippocampus after SE induced by Lithium chloride-pilocarpineObjective: To investigate the pathological features of hippocampal neuronal injury induced by PILO in rat epilepsy model.The changes of autophagy in hippocampus of rats with epilepsy were studied.Methods: Wistar rats were included in the study.Fifty adult male and healthy Wistar rats were randomly divided into 5 groups: control group,3h post-SE group,6h post-SE group,12 h post-SE group,24 h post-SE group.After the model was made,brain slices(5 per group)were subjected to Tunel staining,HE staining and Nissl staining,and tissue samples(5 per group)were used for western blot to detect autophagy-related proteins P62,LC3 and Beclin1.The expression of hippocampal neurons in hippocampus of rats with epilepsy induced by lithium-pilocarpine and the expression of autophagy-related proteins in hippocampus were observed.Results:1.Typical episode of seizure occurred 2-20 min after PILO injection: manifested as blood tears,blinking,moving whiskers,rhythmic chewing or exploring behavior,repeated head and neck ups,followed by facial myoclonus,nodding,unilateral or bilateral forelimbs The clonic,erect,fall,or flip,can eventually progress to a generalized tonic state.In this group of 40 rats,all grade III-V episodes appeared,and finally SE(the survival of 38,2 deaths).Two hours after the onset of SE on the rats,the rats were terminated with diazepam,and the response and activities were reduced.2.Histomorphological results: The neurons in the hippocampal CA1 and CA3 areas of the control group were normal,the cell structure was intact and clear,the arrangement was neat and tight,the nucleus was round or elliptical,the cytoplasm was transparent,the chromatin was evenly distributed,and the nucleolus was distributed.Clear.In the model group(3h,6h,12 h,24h),the neurons in the hippocampal CA1 and CA3 regions were incomplete,arranged disorderly,the cells were swollen and ruptured,the cell spacing was increased,the contour was blurred,the boundary was unclear,and there were a large number of degeneration and necrotic neuron cell bodies.The cell nucleus is concentrated,the nucleus is pyknosis,the nucleolus is not clear,and the normal structure is lost.The 24 h post-SE is most obvious.Nissl staining results: The vertebral cells in the hippocampal CA1 area of the control group were clear and intact,the nuclear structure was normal,the chromatin distribution was uniform,and the Nissl bodies in the cytoplasm were abundant.In the model group(3h,6h,12 h,24h),the histomorphology of hippocampal CA1 and CA3 showed swelling,disordered arrangement,membrane rupture,decreased cytoplasmic Nissl bodies,and the more obvious neuronal death with time,especially at 24 h.3.TUNEL fluorescence: TUNEL-positive cells were not detected in hippocampal staining of control rats.The number of TUNEL-positive cells in hippocampus was expressed in model group(3h,6h,12 h,24h),and 24 was most obvious with prolonged positive cells.4.Western blot analysis showed that the ratio of LC3II/LC3 I and Beclin1 expression in hippocampus of rats at 3h,6h,12 h and 24 h after epilepsy were decreased(n=5,P<0.05),and the expression of p62/SQSTM1 was increased(n=5,P < 0.05).Conclusion: The number of hippocampal neurons in rats with lithium-pilocarpine epilepsy is reduced and damaged,and the autophagy of injured neurons is inhibited.Part Ⅱ Effects of different doses of baicalin on the behavior of rats with epilepsy induced by lithium chloride-pilocarpine and its mechanismObjective: To study the behavioral effects of different doses of baicalin on the status of epilepticus in rats and its mechanism.Methods: Fifty Wistar rats were randomly divided into 5 groups: seizure group(24h post-SE),n=10;baicalin intervention group 4(50mg/kg,100mg/kg,150mg/kg,200mg/kg),n=10 per group.After intraperitoneal injection of pilocarpine,the rats were placed in a 40 cm × 80 cm cage and the animals were closely observed for 2 h and the experimental records were recorded.After the model was successful,brain slices(5 per group)were subjected to Tunel staining,HE staining and Nissl staining,and tissue samples(5 per group)were used for western blot to detect autophagy-related proteins P62,LC3 and Beclin1 to observe the damage of different doses of baicalin on hippocampal neurons in rats with epilepsy induced by lithium-pilocarpine and the expression of autophagy-related proteins in hippocampus.Results: 1.Latent period(the time of seizure above Racine IV): 29.60±6.603 in the epilepsy model group,41.20±11.84 in the baicalin(50mg/kg)intervention group,and 41.70±10.93 in the baicalin(100mg/kg)intervention group.Baicalin(150mg/kg)intervention group 45.70±9.190,baicalin(200mg/kg)intervention group 63.80±11.73;compared with epilepsy model group(24h post-SE),the incubation period of different doses of baicalin intervention group was significantly prolonged,and The difference was statistically significant(P<0.01).There was no significant difference between the different doses of baicalin(50,100,150,200 mg/kg),but the seizure latency of the 200 mg/kg intervention group was significantly prolonged.2.Histomorphological results: The model group(24h post-SE)hippocampal CA1,CA3 area neurons incomplete morphology,disordered arrangement,cell swelling,rupture,increased cell spacing,blurred contours,unclear boundaries,a large number of degeneration,The necrotic neuron cell body,the nucleus is concentrated,the nucleus is pyknosis,the nucleolus is not clear,and the normal structure is lost.The degree of hippocampal neuron damage in different doses of baicalin intervention group was alleviated to some extent.Nissl staining results: the model group(24h post-SE)hippocampal CA1,CA3 area histomorphology showed neuronal swelling,disordered arrangement,membrane rupture,cytoplasmic Nissl bodies decreased,different doses of baicalin intervention group hippocampal neuron damage degree There are different degrees of relief.3.TUNEL fluorescence: The number of TUNEL-positive cells in the hippocampus of the model group(24h post-SE)was expressed.The number of TUNEL-positive neurons in the hippocampus of different doses of baicalin was decreased to different extents.Western blot analysis showed that the ratio of LC3II/LC3 I in hippocampus and the expression of Beclin1 in different concentrations of baicalin(50mg/kg,100mg/kg,150mg/kg,200mg/kg)compared with PILO-induced epileptic model(24h postSignificantly increased in the SE)group(n=5,P<0.05),and the expression of p62/SQSTM1 was significantly decreased(n=5,P<0.05).5.Immunofluorescence results,single-labeled P62/SQSTM1 expression in different concentrations(50mg/kg,100mg/kg,150mg/kg,200mg/kg),the expression of Beclin1 was decreased in different tissues.There are different degrees of elevation.Conclusion: The results show that early application of baicalin can protect hippocampal neurons in seizures;baicalin may exert neuroprotective effects by up-regulating the autophagy activity of damaged neurons.Part Ⅲ: Up-regulation of autophagy activity in hippocampal neurons by PI3-K/Akt/mTOR signaling pathway possibly be one of the mechanisms of baicalin to alleviate brain damage in seizuresObjective: To investigate the expression of autophagy in hippocampal neurons of rats with epilepticus status(24h post-SE)and pretreatment with baicalin(200mg/kg);and to investigate whether baicalin preconditioning up-regulates hippocampal autophagy activity via PI3-K/Akt/mTOR pathway,thereby reducing hippocampal neuronal damage caused by epilepsy.Methods: Twenty rats were divided into 4 groups: control group,24 h post-SE,24 h post-SE+Baicalin,24 h post-SE+Baicalin+3MA(administered lateral ventricle injection of 3-methylindole-selective autophagy).After the successful model,the brain slices(5 per group)were subjected to Tunel staining,HE staining and Nissl staining,and tissue samples(5 per group)were used for western blot detection of autophagy-associated proteins P62 and LC3.,Beclin1,expression of the apoptotic proteins Bcl2,Bax,cleaved-caspase3 and the pathway proteins PI3 K,Akt,p-Akt,mTOR,p-mTOR,ULK1,p-ULK1.Results: 1.Histomorphological results: The neurons in the hippocampal CA1 and CA3 areas of the control group were normal,the cell structure was intact and clear,the arrangement was neat and tight,the nucleus was round or elliptical,the cytoplasm was transparent,and the chromatin was Evenly distributed,the nucleolus is clear.In the model group(24h post-SE),the neurons in the hippocampal CA1 and CA3 regions were incomplete,arranged disorderly,the cells were swollen and ruptured,the cell spacing was increased,the contour was blurred,the boundary was unclear,and there were a large number of degenerated and necrotic neurons,and the nuclei were concentrated.The nucleus is pyknosis,the nucleolus is not clear,and the normal structure is lost.The degree of hippocampal neuronal damage in the baicalin intervention group(SE+B)was alleviated to varying degrees.Compared with the baicalin intervention group,the 3-MA intervention group(SE+B+3-MA)increased the number of morphological abnormal neurons in the hippocampal CA1 and CA3 areas,increased cell swelling,and even ruptured,increased cell spacing,blurred contours,and boundaries.Unclear,degeneration,necrosis,neuronal cell body enlargement,nuclear condensation,nuclear pyknosis,nucleoli are not clear,lose normal structure.Nissl staining results: The vertebral cells in the hippocampal CA1 area of the control group were clear and intact,the nuclear structure was normal,the chromatin distribution was uniform,and the Nissl bodies in the cytoplasm were abundant.The histomorphology of hippocampal CA1 and CA3 in the model group(24h post-SE)showed swelling and disorder of the neurons,membrane rupture,and decreased cytoplasmic Nissl bodies.The degree of hippocampal neuronal damage in the baicalin intervention group was alleviated to varying degrees.Compared with the baicalin intervention group,the 3-MA intervention group(SE+B+3-MA)had increased swelling and even rupture of the neurons in the hippocampal CA1 and CA3 areas,and the cytoplasmic Nissl bodies were reduced.2.TUNEL fluorescence: TUNEL-positive cells were not stained in the hippocampal CA1 region of the control rats.The number of TUNEL positive cells in the hippocampus of the model group(24h post-SE)was expressed.The number of hippocampal neuronal apoptosis in the baicalin intervention group was significantly reduced.Compared with the baicalin intervention group,the number of TUNEL-positive neurons in the hippocampus was significantly increased in the 3-MA intervention group(SE+B+3-MA).3.Western blot: Compared with the model group(24h post-SE),the 3-MA intervention(SE+B+3-MA)group showed a significant decrease in LC3 II and Beclin 1 and an increase in the protein level of P62.Compared with the control group(Control),the levels of Bax and cleaved-caspase3 in the neurons of the model group(SE)group were increased,and the protein level of Bcl-2 was significantly decreased.The Bax and cleaved-caspase3 protein levels were significantly decreased after baicalin intervention,and the expression of Bcl-2 protein was significantly increased.The 3-MA intervention group(SE+B+3MA)significantly enhanced Bax and decreased Bcl-2.Compared with the control group,the expression of p-AKT,p-mTOR and p-ULK1 was significantly increased in the model group,and the expression was significantly decreased in the baicalin intervention group.Increased expression of p-AKT,p-mTOR,and p-ULK1 in the 3-MA intervention groupConclusion: Epileptic status leads to decreased autophagy activity,neuronal necrosis and apoptosis in hippocampal neurons;baicalin preconditioning up-regulates hippocampal autophagy activity through PI3-K/Akt/mTOR signaling pathway,which possibly be baicalin preconditioning to reduce epilepsy one of the mechanisms of hippocampal damage. |
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