Neuroprotective Effects Of Pabing 2 Formual In Dopaminergic (DA) Neurons Under Oxidative Stress Damage

Objective1.To investigate the neuroprotective effects of Pabing 2 Formual on PD rats.2.To research the neuroprotective effects of Pabing 2 Formula' s medicated serum on Dopamine neurons induced by iPSC through lowering ROS.3.To research the protection of Pabing 2 Formual on the translated DA cells induced by iPSC of the PD mice.MethodsThe preparation of PD rat model,lavage and related indicators of detection.1.6-OHDA were injected into striatum of Wistar rats to construct the PD rat model;the neck and back were injected with APO by subcutaneous regularly,observing whether rotation behavior.The model is successful if the number of rotation is more than 210 r/30min,then given different doses of the Pabing 2 liquid,and observed the changes of rotating within four weeks.2.Picking up some fresh brain tissue,fixing with 10%formaldehyde,embedding with dehydratio,tissue section,and detecting the expression of PD rats' TH by using immunohistochemical staining method.3.Taking part of the fresh brain tissue,extracting total protein,and using Western Blots method to detect the expression of PD rats' Nrf2 protein.4.Taking part of the fresh brain tissue,extracting RNA,and using qPCR method to detect the expression of PD rats' antioxidant related gene NQO1,HO-1,MRP2,GPX2.The establishment of cell injury model,medicated serum intervened and related index detection.1.DA cells were intervened with 10%serum for 12h in advance,then model group and treatment group were damaged with 100?M H2O2 for 12h respectively,and blank group was added equivalent cell culture medium.Finally,we collected the cells to observe the levels of ROS in cells by adopting active oxygen detection kit for streaming.2.Collected the cells,and used immunofluorescence staining method to detect the positive rate of TH,TUJ-1,and TH/TUJ-1 double positive rate in cells.3.Collected cells,extracted RNA,reverse transcription,and used qPCR method to detect antioxidant related gene Nrf2,NQ01,HO-1,MRP2,GPX2 expression in DA cells.4.Collected cells,extracted RNA,reverse transcription,and used qPCR method to detect DA related gene Nurrl,TH,TUJ-1,PITX3,Foxa2 expression.Preparation of PD mice model,transplantating DA cells,lavage and related index detection.1.NOD scid mice were injected with MPTP(40 mg/kg)to build the PD mice model,and translating 1X105 DA cells induced by iPSC cells into striatum,observing the change of behavior within 4 weeks.2.Collecting animal materials,taking fresh midbrain organization,extracting RNA,reverse transcription,qPCR method was used to detect the expression of TH.ResultsThe neuroprotective effects of Pabing 2 Formual on PD rats.1.Effect of Pabing 2 Formual on PD rats' behavioral:the sham group which was induced by APO did not appear abnormal rotation behavior,but the PD rats which were injected with 6-OHDA showed abnormal rotation behavior after induced by APO,and the rotation number was more than 210r for 30min.In the third week,the rotation speed of high and middle dose group was reduced,and the number of rotation was lower than 210r for 30min.Compared with model group,the difference was statistically significant.In the fourth weeks,the rotation speed reached the lowest in the high and middel dose group,and compared with model group,there were significant differences.2.Effect of Pabing 2 Formual on PD rats' TH:the level of TH was higher in the high dose group compared with model group,and there were significant differences.3.Effect of Pabing 2 Formual on PD rats' Nrf2 protein expression:the high dose group of Nrf2 protein expressed higher than model group,and there was significant difference.4.Effect of Pabing 2 Formual on PD rats' antioxidant related genes expression as NQO1,HO-1,MRP2,GPX2:compared with model group,the expression of NQO1 and GPX2 gene in the high dose group increased,and the difference was statistically significant.Compared with model group,the expression of MRP2 and HO-1 gene in the high dose group increased,and there was significant difference.The neuroprotective effects of Pabing 2 Formula' medicated serum on Dopamine neurons induced by iPSC through lowering ROS.1.ROS levels:the level of ROS in the model group was higher than medicated group and control group,and the difference between groups was significant.2.Immunofluorescence staining:the TH/TUJ-1 positive cell numbers in medicated serum group was higher than model group,and there were significant differences.3.The antioxidant related gene expression:compared with model group,the expression of antioxidant genes NQO1 and HO-1 is higher in the medicated serum group,there are significant difference;also the expression of antioxidant genes MRP2 and GPX2 is higher in the medicated serum group compared with model group,the difference was statistically significant.4.The DA related gene expression:the expression of TH,TUJ-1,PITX3 and Foxa2 in the medicated serum group was higher than model group,and the difference was statistically significant.The protection of pabing 2 Formual on the translated DA cells induced by iPSC of the PD mice.1.Behavioral detection:the incubation period in treatment group was longer than the untreated group,and the difference was statistically significant.The number of dropping in the treatment group was less than that in the untreatment group,and the difference was statistically significant.2.Gene expression:the gene expression of TH in the treatment group was higher than the untreated group,and the difference was statistically significant.Conclusion1.Pabing 2 Formula can reduce the number of rotation and adjust the level of TH for PD models,and restrain the ROS result from the neurotoxic substance to the brain,thus protecting the DA neurons through enhancing the expression of Nrf2 gene and protein together with other related anti-oxidation factor.2.The DA cells were treated with medicated serum of Pabing 2 Formula after damaged by H2O2,which can result in enhancing the gene expression of NOQ1 and other related anti-oxidation factors,also increasing the gene expression of TH and other related dopaminergic neurons,thus protecting the DA neurons.3.Pabing 2 Formula can prolong the transplated DA neurons' survival cycle.