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The mechanism of PTEN nuclear translocation induced by hypoxia ischemia and the neuroprotective effect of inhibited PTEN nuclear translocation in treatment of HIBD in neonatal ratsObjective:Hypoxic-ischemic encephalopathy?HIE?is a major cause of mortality and severe neurologic impairment in neonatal or perinatal.However,few satisfactory therapeutic strategies are available.Our recent study has found that the nuclear accumulation of phosphatase and tensin homolog deleted on chromosome ten?PTEN?was increased in in vitro and in vivo models of excitotoxic/ischemic neuronal injuries,and prevention of PTEN nuclear translocation reduced the neuronal death induced by excitotoxicity/ischemia.In the present study,we aim to explore the potential mechanism of PTEN nuclear translocation and the neuroprotective effect of inhibited PTEN nuclear translocation on neuronal injury induced by hypoxia ischemia in developing brain,and elucidate the protective effect of inhibited PTEN nuclear entry on behavioral impairment in hypoxic-ischemic brain damage?HIBD?rats,the model of HIE.Methods:?1?Pregnant Sprague-Dawley?SD?rats?E18-20?were used in the present study to culture cortical/hippocampal neurons.Three days later,the primary neurons treated with lentivirus.Seven days later,the primary neurons were pre-treated with peptide Tat-K13 and control peptide Tat-K13R for 1h,and then subjected to oxygen-glucose deprivation?OGD?:the cell conditioned medium was replaced by sugar-free EBSS and treated with 5%O2+95%N2.The CTR group without OGD treatment.The levels of PTEN,neural precursor cell expressed developmentally down regulated gene 4-1?NEDD4-1?,NEDD4-2 protein expressions in total protein and the level of PTEN protein in nuclear protein were detected by western blot at different time points?3h,6h,9h,12h,24h?after OGD.PTEN nuclear translocation visualization was detected by immunofluorescence.The levels of PTEN protein expression in total and nuclear protein were detected by western blot.And LDH assay,MTT assay and CCK-8 assay were used to detect the cells death and viability at 6h after OGD.Co-lmmunoprecipitation experiment was performed to determine the interaction of PTEN and NEDD4-1 or NEDD4-2.Ubiquitination western blot was used to detect the mono-uibiquitination of PTEN both in HEK293T cells transfected with wild-type or mutant PTEN plasmid and in neurons infected with wild-type or mutant PTEN lenti virus at 6h after OGD.The levels of NEDD4-1,NEDD4-2 protein expression in total protein and the level of PTEN protein in nuclear protein were detected by western blot in primary neurons infected with interference or overexpression of NEDD4-1 or NEDD4-2 lentivirus at 6h after OGD.?2?7-day-old SD rats were divided into two groups of the sham operation group?sham group?and the operation group?HIBD group?,randomly.Rats in HIBD group were ligated the left common carotid artery according to the method Rice,and then exposed to hypoxic conditions?8%O2+92%N2?to establish HIBD model.The rats in HIBD were randomly divided into saline treatment group?HIBD+saline?,peptide Tat-K13 treatment group?HIBD+K13?and control peptide Tat-K13R treatment group?HIBD+K13R?.The rats in HIBD+K13 and HIBD+K13R group received intraperitoneal?i.p.?injection of Tat-K 13 and Tat-K13R,respectively.The first and second bolus of peptide were administered at 0 h and 3 h after operation,respectively,and another two doses of peptide were conducted on the second and third days,respectively.The levels of PTEN protein expressin in total protein and in nuclear protein were detected by western blot at 6h after HIBD model established.Four weeks after HIBD model established,the Morris water maze test was performed to measure spatial learning and memory retrieval ability.Results:?1?Western blot results showed that NEDD4-1 protein expression level was significantly decreased at 3-9 h?p<0.05?and reached a trough at 6-9h?p<0.01?,but NEDD4-2 protein expression level was significantly increased at 6-9 h?p<0.05?and reached a peak at 9h?p<0.01?,also both of them returned to control levels within 12 h,after OGD in total protein in primary neurons.Nuclear PTEN levels significantly increased at 3-12h?p<0.01?and reached a peak at 6-9h after OGD,returned to control levels within 24 h,while no significant difference of total PTEN protein expression level was found in each group in primary neurons.?2?Immunofluorescence results showed that under normal circumstance,PTEN was predominantly found in the cytoplasm in primary neurons.Wliile,the subcellular localization of PTEN was from the cytoplasm to the nucleus after OGD,gradually.The enhanced PTEN nuclear translocation was time-dependent,and PTEN was mainly localized in cytoplasm within 24 hours after OGD.?3?Western blot revealed that mono-ubiquitinated PTEN was appeared in OGD group both in HEK293T cells and in neurons,and mono-ubiquitinated PTEN decreased and nucleus PTEN significantly reduced?p<0.01?in the case K13R mutation compared to wild PTEN group;while,there were no significant difference of mono-ubiquitinated PTEN and nucleus PTEN in the case K289R mutation compared to wild PTEN group.?4?Western blot results showed that LVshNEDD4-1 or LVNEDD4-1-treated neurons displayed no appreciate effect on PTEN nuclear translocation,however,NEDD4-1 over-expression before OGD significantly blocked PTEN nuclear import induced by OGD in primary neurons?p<0.05?,the nucleus PTEN in LVNEDD4-1+OGD group displayed no significant difference compared with CTR group:and no mono-ubiquitinated PTEN was detected in LVnedd4-i+OGD group.?5?Western blot results also showed that treatment with LVshNEDD4-2 or LVNEDD4-2 had no appreciate effect on PTEN nuclear import in primary neurons with or without OGD.?6?CO-IP results showed both NEDD4-1 and PTEN could pull down each other in HEK293T cells and primary neurons,while NEDD4-2 and PTEN couldn,t mutually coprecipitate each other.?7?Compared with CTR group,the level of PTEN in nuclear was no significant change,the LDH release was significantly increased?p<0.05?,and the cell viability was significantly decreased?p<0.05?in neurons pre-treatment with Tat-K13 before OGD;however,compared with OGD group,the level of PTEN in nuclear was significantly decreased?P<0.01?,the LDH release was significantly decreased?P<0.001?,and the cell viability was significantly increased?p<0.01?in neurons pre-treatment with Tat-K13 before OGD.Moreover,compared with CTR group,the level of PTEN in nuclear was significantly increased?p<0.01?,the LDH release was significantly increased?P<0.001?,and the cell viability was significantly decreased?p<0.001?in neurons pre-treatment with Tat-K13R before OGD.?8?Systemic administration of Tat-K13 induced a dose-dependent decrease in PTEN nuclear translocation?p<0.05?,while administration of Tat-K13R displayed no effect on PTEN nuclear translocation induced by HIBD.Morris water maze data demonstrated that the escape latency of HIBD+K13 group rats was significantly increased compared with rats in sham group?p<0.05?,and was obviously decreased compared with rats in HIBD+saline group?P<0.01?during spatial training period;however,the escape latency of HIBD+K13R group rats was significantly increased compared with rats in sham group?p<0.01?.The results from probe test demonstrated that compared with HIBD+saline group,rats treatment with Tat-K13 spent much more time in the hidden platform-located quadrant?p<0.01?,and the number of crossing the location of hidden platform was obviously increased?P<0.01?;while,control peptide Tat-K13R existed no effect on the time spent in the hidden platform-located quadrant and the number of entries into the platform zoneConclusions:?1?The enhanced PTEN nuclear translocation is associated with PTEN mono-ubiquitination,and the K13 residue of PTEN is critically important for OGD-induced mono-ubiquitinated PTEN nuclear translocation,but K289 residue of PTEN display no effect on OGD-induced mono-ubiquitinated PTEN nuclear translocation.?2?NEDD4-1 over-expression before OGD could block PTEN mono-ubiquitination induced by OGD,and then significantly suppress PTEN nuclear import induced by OGD,however,downregulation of NEDD4-2 before OGD display no appreciate effect on PTEN nuclear translocation induced by OGD in primary neurons And only regulate NEDD4-1 or NEDD4-2 also display no appreciate effect on PTEN nuclear import.?3?There is an interaction between PTEN and NEDD4-1,but not NEDD4-2.?4?Tat-K13 could suppress PTEN nuclear translocation induced by OGD,and then suppress the neurons injuries induced by OGD,thereby ameliorates behavioral defects induced by HIBDPart ? Tanshinone I alleviates motor and cognitive impairments via suppressing PTEN nuclear translocation induced by oxidative stress in the neonatal rats after HIBDObjective:Neonatal hypoxia-ischemia is one of the major reasons that cause neuronal damage and neonatal death.There is mounting evidence that tanshinone I?TsI?,one of the extracts of Danshen,displays potential neuroprotective effect in adult mice subjected to ischemia,the potential mechanism may be associtated to the antioxidantive effect of TsI.Another study identified that PTEN nuclear accumulation induced by oxidative stress supressed the growth of cancer cells.However,it is unclear whether treatment of TsI has neuroprotective effect on neonatal hypoxic-ischemic brain damage?HIBD?,and if so,the potential mechanisms also remain unknow.In the present study,we aim to explore the underlying mechanism of neuroprotective effect of TsI on neuronal injury and behavioral impairment induced by hypoxia ischemia in HIBD rats,and explore the potential mechanismMethods:?1?7-day-old SD rats were divided into the two groups of the sham group and HIBD group,randomly.The rats in HIBD group were randomly divided into saline treatment group?HIBD+saline?and TsI treatment group?HIBD+TsI?.The rats in sham+TsI and HIBD+TsI groups received intraperitoneal?i.p.?injection of TsI at a dose of 5 mg/kg/day for 7 days,while the rats in sham and HIBD+saline groups only received intraperitoneal?i.p.?injection of the same volume of sterile saline.Left brain tissues of hippocampus and cortex collected at 48h after HIBD model established in ice to extract protein for detecting the antioxidant activity?total antioxidant capacity,glutathione,catalase and total superoxide dismutase?and pro-oxidants?hydrogen peroxide and nitric oxide synthase?by using ELISA kit.Three weeks after HIBD model established,grasping test and rotarod test were used to evaluate myodynamia and motor performance,respectively.Four weeks after HIBD model established,the Morris water maze test was performed to measure spatial learning and memory retrieval ability.After behavioral tests,the rats were perfused with 0.9%saline followed by 4%paraformaldehyde in 100 mM phosphate-buffered saline,and immunohistochemistry was conducted to detect the number of neurons of the rats.The primary neurons were pre-treated with TsI for lh?and then subjected by OGD treatment.The level of PTEN in nuclear protein was detected by western blot at 6h after OGD.Results:?1?Data of grasping test showed that myodynamia of the right forelimb was significantly decreased compared to that of the left in HIBD+saline rats?p<0.05?,while the myodynamia of right forelimb was similar to the left in the rat treatment with TsI.?2?Rotarod test results showed that compared with sham group,the rats in HIBD+saline group spent much less time on the rod?p<0.05?,while the time spent on the rod was dramaticaly increased in HIBD+TsI rats compared to HIBD+saline group rats?p<0.05?.?3?Morris water maze data demonstrated that the escape latency in the HIBD+saline group was much longer than that in the sham group during spatial training period?p<0.01?,while the latency to the platform in the rats treatment with TsI before or after HIBD were obviously decreased compared with saline treatment?p<0.05?.The results form probe test demonstrated compared with sham group,rats in HIBD+saline group spent much less time in the quadrant where the hidden platform was previously located?p<0.01?,the escape latency to cross the location of hidden platform was much longer?p<0.01?5 the number of crossing the location of hidden platform was significantly decreased?p<0.01?.Although the time spent in the hidden platform-located quadrant of the rats treatment with TsI after HIBD displayed no signifcant difference compared to the rats in HIBD+saline,rats treatment with TsI before HIBD spent much more time in the hidden platform-located quadrant?p<0.05?.And the escape latency to cross the location of hidden platform was much shorter?p<0.05?,the number of crossing the location of hidden platform was obviously increased?p<0.05?in the rats treatment with TsI before or after HIBD,compared to the rats in HIBD+saline group.?4?Immunohistochemistry results showed that compared with sham group,the number of NeuN-immunoreactive neurons in the CA1 area dramatically decreased in the HIBD group?p<0.01?,and the number of NeuN-immunoreactive neurons of rats in HIBD+TsI group was obviously increased compared with HIBD+saline group?p<0.01?.?5?ELISA results demonstrated that a significant decrease in the production of antioxidants?T-AOC,GSH?CAT and T-SOD??p<0.05?and a significant increase in the production of pro-oxidants?H2O2?TNOS and iNOS??p<0.01?were observed in HIBD+saline group compared with sham group.However,compared with saline treatment,the production of antioxidants of rats in TsI treatment group was significantly increased?p<0.05?,and the production of pro-oxidants of rats in TsI treatment group was significantly decreased?p<0.05?.?6?Western blot results showed that nucleus PTEN was significantly increased after OGD in primary neurons,while compared with OGD group?m<0.01?,the nucleus PTEN was significantly decreased in neurons pre-treatment with TsI?p<0.05?.Conclusions:These data of present study provide the first evidence that administration of TsI suppresses HIBD-induced neuronal death and oxidative stress,thereby suppresses PTEN nuclear translocation,and then ameliorates myodynamia and motor abilities as well as spatial learning and memory in neonatal rats after HIBD,an animal model of HIE in patient,suggesting that TsI may represent a potentially effective therapeutic drug for HIE. |
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