Neuroprotective Effect And Mechanism Of Tangeretin In Rats With Seizures Induced By Lithium-pilocarpine

Objective:Epilepsy is one of the most common and serious neurological diseases.Epilepsyis a neurological disorder characterized by epileptic seizures as a result of abnormal electrical discharge in the brain.The major population who suffered from epilepsy is children and the elderly.It has been recorded that about 50%of epilepsyoccurs in children and the elderly,and half of them occurs in infants under one year of age.In 2015,about 39 million people around the world were diagnosed with epilepsy and 12 thousand and 500 people died of it.At present,the treatment of the disease mainly focuses on drug control of epileptic seizure and assisted by the management of diet.To date,there is no antiepileptics has been found to be both effective and totally safety,that without any side-effects on intelligence,excitability,motor function,mood or other aspects.Considering about the limitation of the traditional antiepileptics,and its side-effects,novel antiepileptic drugs with more effective effects and lower side-effects are still required to be developed.Traditional Chinese medicine has been considered as a kind of mild and safe treating-choose.Traditional Chinese medicine is extracted or synthesized from natural herb plants.In China,treating epilepsy with traditionalChinese medicine has a long history.There are 23 kinds of Chinese herbal medicines with anti-epileptic effect have been recorded in Chinese Pharmacopoeia,includingQingyang ginseng,Scutellaria baicalensis,Gastrodia elata,Radix bupleuri,Coptis chinensis,etc.Tangeretin(C20H20O7,molecular weight372.37)is an o-type polymethoxyflavone.It is mainly existed in the peel of Citrus nobilis Lour.and C.aurantium L.,as well as in the leaves and stems of C.reticulata Blanco.The multiple biological activities of Tangeretin have been widely-reported,including increasing insulin secretion,regulating the activity of liver enzymes,and reducing glucose induced by Streptococcus.Besides,Tangeretin exerts significant anti-inflammatory and antioxidant functions,and protective effects on cardiovascular and kidney.Recently,several researchers found that Tangeretin also exhibited neuroprotective effect.It has potential in preventing or treating Alzheimer's and Parkinson's.However,to our best of knowledge,little was known regarding the effect of Tangeretin on epilepsy.The present work studied the effects of Tangeretin on the severity of seizures and hippocampal neuronal damage in epileptic rats.Meanwhile,we measured the expression changes of apoptosis-relatedproteins and matrix proteins in neurons.This study aimed to reveal the neuroprotective effect of Tangeretin,and its underlying mechanisms.The findings of this study will provide a theoretical basis for elucidating the antiepileptic mechanism of Tangeretin.Methods:Part One,Effects of Tangeretin on progression of seizure activityand hippocampal neuronal damage in epileptic ratsA total of 30 adultWistar rats(clean grade,all male)were enrolled in this study and were randomly divided into five groups(n =6 per group):Control group,Pilocarpine(PL)group,PL + 50 mg Tangeretin group,PL + 100 mg Tangeretin group,and PL+ 200 mg Tangeretin group.The rats in PL group receivedintraperitoneal(i.p.)injection of 3 mmol/kg lithium chloride(LiCL),and 24 h later subcutaneous(s.c.)injection of 1 mg/kgscopolamine hydrobromide(to prevent peripheral cholinergic response induced by pilocarpine),and 30 min later i.p.injection of 30 mg/mL pilocarpine.The rats in Tangeretin-treating groups were i.p.injected with 3 mmol/kg LiCL,24 h later i.p.injected with 50,100 or 200 mg Tangeretin,30 min later s.c.injected with 1 mg/kgscopolamine hydrobromide,and 30 mim later i.p.injected with 30 mg/mL pilocarpine.After the indicated administration,hippocampal tissues were removed from rats,and were dehydrated,paraffin embedded and cut into slices for use in Nissl staining and terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL).Part Two,Tangeretin inhibits neuronal apoptosis in epileptic rats induced by pilocarpine through regulating apoptosis-related protein and PI3K/AKT pathwayAfter the indicated administration,the total protein in hippocampal tissues was extracted by using RIPA lysis buffer.Meanwhile,nucleoprotein extraction kit and mitochondrial protein extraction kit were respectively used to extract the protein in nuclei and mitochondria.Western blot analysis was conducted to assess the protein levels of apoptosis-related factors and core factors in PI3K/AKT signaling pathway.Part Three,Tangeretin inhibits the expression of MMP-2 and MMP-9 in epileptic ratsTrizol reagent was used to isolate the total RNA in hippocampal tissues.qRT-PCR was performed to measure the mRNA levels of MMP-2 and MMP-9.Meanwhile,western blot and Gel zymography were used to test the protein levels and the activity of MMP-2 and MMP-9.Results:Part One,Effects of Tangeretin on progression of seizure activityand hippocampal neuronal damage in epileptic ratsNo epileptic seizure symptoms were observed in Control group of rats,but different degrees of epileptic seizure symptoms were found in other four groups of rats.Compared with PL group,Tangeretin pretreatment remarkably reduced the progression of seizure activity.The first period of seizures occurred at 11.02±1.50 min in PL group,and Tangeretin treatment significantly prolonged the latent period,as the first period of seizures in PL + 50 mg Tangeretin,PL + 100 mg Tangeretin and PL+ 200 mg Tangeretin groups were respectively 18.15±1.45 min,25.2±2.05 min and 33.5±1.20 min.The latency of IV grade occurred at34.10±1.50 min in PL group,while Tangeretin pretreatment also could significantly prolong the latent period of IV grade.The latency of IV grade occurred at41.15±1.25 min,48.20±2.05 min and 50.5±1.20 min respectively in PL + 50 mg Tangeretin,PL + 100 mg Tangeretin and PL + 200 mg Tangeretin groups(p<0.05).Nissl staining results showed that,neurons in the hippocampal CA3 region presented string distribution,cells arranged closely,and the structure was clear with regular spherical or elliptical shapes.The PL-treated neurons exhibited an obvious damage situation that intercellular space increased,arrangement was lost,cells became bigger,cell structure was unclear,and cell soma was atrophy.However,Tangeretin pretreatment could significantly attenuate the damage induced by PL.Besides,we found that theviable cell counts in the CA3 hippocampal regions were significantly decreased in PL group,as compared to those in Control group.And also,theviable cell counts were significantly increased in Tangeretin pretreated group than in PL group(p<0.05).TUNEL assay results showed that,TUNEL-positive cells in the CA3 hippocampal regions were significantly increased in PL group,as compared to Control group.As expected,theTUNEL-positive cells were significantly decreased in Tangeretin pretreated group than in PL group(p<0.05),and the increase was does-dependent.Part Two,Tangeretin inhibits neuronal apoptosis in epileptic rats induced by pilocarpine by regulating apoptosis-related protein and PI3K/AKT pathwayWestern blot results showed that,the protein levels of Bad,Bax and Cleaved-caspase-3 were remarkably up-regulated,while Bcl-2 and Bcl-xL were remarkably down-regulated in PL group,as compared to Control group(p<0.05).Tangeretin pretreatment significantly up-regulated Bcl-2 and Bcl-xL protein levels,and significantly down-regulated Bad,Bax and Cleaved-caspase-3 protein levels(p<0.05)when compared to PL group.It seems that Tangeretin could impact the expression levels of apoptosis-related proteins,and the regulatory impacts were in a dose-dependent fashion.The protein levels of apoptosis-inducing factor(AIF)in the nucleus of the hippocampus in PL group were much higher than those in Control group,but the protein levels of AIF in mitochondria in PL group were much lower than those in Control group.Tangeretin pretreatment decreased the protein levels of AIF in the nucleus of the hippocampus,and increased the protein levels of AIF in mitochondria of the hippocampus,as compared to PL group.PL + 200 mg Tangeretin group showed the most remarkable effects on PL-induced alterations of AIF expression.Phosphorylation levels of AKT and GSK-3? were decreased,while protein level of phatase and tensin homology deleted on chromosome 10(PTEN)was increased in PL group compared to Control group.Besides,the protein levels of downstream factors of AKT(mTORc1 and cyclinD1)were significantly down-regulated in PL group(p<0.05).Tangeretin pretreatment significantly induced the phosphorylation of AKT and GSK-3(3,up-regulations of mTORcl and cyclinDl proteins,and down-regulation of PTEN protein(p<0.05).The regulation of Tangeretin pretreatment on these protein expressions were also in a dose-dependent manner.Part Three,Tangeretin inhibits the expression of MMP-2 and MMP-9 in epileptic ratsqRT-PCR results showed that,after 12 h of PL injection,the mRNA levels of MMP-2 and MMP-9 were unchanged in PL group when compared to Control group.Also,mRNA levels of MMP-2 were unaffected by Tangeretin pretreatment,but mRNA levels of MMP-9 could be remarkably increased by Tangeretin pretreatment.After 24 h of PL injection,the mRNA levels of MMP-2 and MMP-9 were significantly increased in PL group,as compared to Control group.However,no significant differences between PL group and Tangeretin pretreated groups were observed in mRNA levels of MMP-2 and MMP-9.Western blot results showed that,after 12 h of PL injection,the protein levels of MMP-2 and MMP-9 were increased,and Tangeretin pretreatment significantly reduced these increases induced by PL.After 24 h of PL injection,same trend was observed.Besides,it seems that the impact of PL on the expression of MMP-2 and MMP-9 is in a time-dependent manner.Gel zymography results showed that,after 12 h of PL injection,the activity of MMP-2 and MMP-9 was significantly increased(p<0.05).Compared to PL group,Tangeretin pretreatment significantly reduced the activity of MMP-2 and MMP-9(p<0.05).After 24 h of PL injection,the same trend was observed.Conclusions:1.Tangeretin can effectively delay the latent period of epileptic seizure and reduce the mortalityof epileptic rats;2.Tangeretin attenuates pilocarpine-induced hippocampal neurons damage,and inhibits hippocampal apoptosis;3.Tangeretin protects hippocampal neurons through reducing AIF nuclear translocation,and activating PI3K/AKT signaling pathway;4.Tangeretin represses the expression and activity of MMP-2 and MMP-9 in pilocarpine-induced seizures.