| Objective:Experiments in vivo and in vitro was used to study medicine food homology of Puerarin in the traditional Chinese herb radix puerariae lobata(Puerarin,PUE)activated PI3K/Akt/Nrf2 signaling pathway inhibition of oxidative stress on cerebral ischemia reperfusion function injury(CIRI),rats had protection Function as well as to the oxygen Deprivation/sugar after sugar after oxygen(Oxygen and Glucose-Deprivation/Reoxygenation,OGD/R)injury protective effects and mechanism of neurons HT22 cells,revealed the medicinal value of puerarin.It provides theoretical basis for the development of TCM with the same origin of medicine and food and the development of TCM health industry.Methods:1.PUE alleviated brain injury after MCAO/R(1)The rat model of middle cerebral artery occlusion and reperfusion(Middle Cerebral Artery Occlusion and Reperfusion,MCAO/R)was established by modified thread occlusion method;(2)The neurobehavioral score of Imax R rats was detected by Longa five-grade score method;then the infarct volume of rats was measured by 2,3,5-triphenyltetrazolium chloride(TTC);(3)The morphological changes of hippocampal neurons on the ischemic side were observed by hematoxylin-eosin(HE)staining;(4)The apoptosis rate of hippocampal neurons was detected by in situ terminal transferase labeling(Tunel-Neu N)staining;(5)The rat model of CIRI was established by MCAO/R,PI3 K inhibitor was given,and the content of ROS in hippocampal neurons in ischemic area was detected by biochemical kit;(6)The contents of superoxide dismutase(SOD),malondialdehyde(MDA),catalase(CAT),glutathione peroxidase(GSH-px),glutathione(GSH)and glutathione peroxidase(GSH-px)were detected by biochemical method;(7)The expression of Nrf2 was detected by immunohistochemistry.(8)The expression of pathway-related proteins PI3 K,p-PI3 K,Akt,p-Akt,Keap1,Nrf2 Nucleus,Nrf2 Cytoplasm and HO-1 in rat hippocampal neurons was detected by Western Blotting.2.Puerarin attenuates hippocampal neuron injury after OGD/R by inhibiting oxidative stress through PI3K/Akt/Nrf2 pathway(1)Normal cultured HT22 cells were taken and observed under light microscope for routine morphology;(2)Oxygen-glucose deprivation and Reperfusion(OGD/R)model was established by replacing complete medium containing serum with sugar-free medium combined with hypoxic chambers;(3)Cell Counting Kit-8(CCK-8)to detect the cell survival rate of HT22;(4)Layered Double Hydroxide(LDH)to detect the LDH release in each group of OGD/R injury;(5)Hoechst 33258 staining to observe the apoptosis of HT22 cells in each group;(6)AVPI flow cytometry to detect the apoptosis of HT22 cells;(6)AV-PI flow cytometry to detect the apoptosis rate of HT22 cells;(7)To verify whether PUE activates PI3K/Akt/Nrf2 pathway to attenuate hippocampal neuronal injury after OGD/R,we established an OGD/R model and administered PI3 K inhibitor;(8)Flow cytometry to detect changes in ROS content in HT22 cells after OGD/R;(9)Biochemical assays for superoxide dismutase(SOD),malondialdehyde(MDA),catalase(CAT),glutathione(GSH)and glutathione peroxidase(GSH-px);(10)Immunofluorescence assay for Nrf2 nucleus entry;(11)Western Blotting assay for pathway-related proteins PI3 K,p-PI3 K,Akt,p-Akt,Keap1,Nucleus Nrf2,and Nucleus Nucleus in rat hippocampal neurons.Nucleus Nrf2,Cytoplasm Nrf2,HO-1;(12)Molecular docking to explore the docking of gerberoside and PI3K/Akt;(13)Drug affinity responsive target stability(DARTS)to verify the binding of PUE to(14)Cellular Thermal Shift Assay(CETSA)to verify the binding of PUE to PI3K;(15)Immunoprecipitation to verify the interaction between Keap1 and Nrf2.Result:1.Puerarin protects cerebral ischemia-reperfusion injury in rats by inhibiting oxidative stress(1)TTC staining and neurobehavioral score showed that compared with the model group,the cerebral infarct volume and neurobehavioral score of rats in PUE administration group were significantly decreased;To explore the mechanism of PUE in reducing ischemic stroke based on PI3K/Akt/Nrf2 inhibition of oxidative stress.(2)The results of HE staining showed that the cells in the hippocampal neurons of the sham operation group were neatly arranged,and the nucleoli in the cells were clear.After MCAO/R modeling,the nucleoli of nerve cells in the hippocampus disappeared,and some nuclei and plasmas contracted and arranged disordered.After PUE treatment,the morphology of cells in this area was improved and tended to the normal level.(3)Tunel-Neu N staining showed that PUE treatment could significantly reduce the apoptosis rate of hippocampal neurons in MCAO/R rats.(4)Compared with the sham operation group,the activity levels of ROS and MDA in MCAO/R group increased,while the activity levels of SOD,CAT,GSH,and GSH-Px decreased;Compared with MCAO/R group,the activity levels of ROS and MDA in LY294002 group increased,while the activity levels of SOD,CAT,GSH and GSH-Px decreased,which were reversed and tended to normal after PUE treatment.(5)Immunohistochemical results showed that when PI3 K inhibitor was given,Nrf2 expression in PUE treatment group was increased compared with the model group;(6)Western Blotting showed that compared with the sham-operated group,the expressions of p-PI3 K,p-Akt,Cytoplasm Nrf2 and Keap1 were decreased in the MCAO/R group,while the expressions of Nucleus Nrf2 and HO-1 were increased.Compared with MCAO/R group,the expressions of Cytoplasm Nrf2 and Keap1 in LY294002 group increased,while the expressions of p-PI3 K,p-Akt,Nucleus Nrf2 and HO-1 decreased.PUE treatment group can reverse the above situation.2.Puerarin attenuates hippocampal neuron injury after OGD/R by inhibiting oxidative stress through PI3K/Akt/Nrf2 pathway(1)The results of CCK8 method and LDH kit showed that the injury degree of HT22 cells was aggravated with the increase of reoxygenation time,and the injury degree reached the peak at 12 h.(2)CCK8 results showed that compared with the Control group,the cell viability of OGD/R group decreased to about 50%;After administration,HT22 cell viability was significantly increased,and cell injury was improved.(3)Under the inverted microscope,HT22 cells in the OGD/R group were shrunken and round,synapses became shorter,adherent and easily floated,while HT22 cells in the PUE administration group were spindle-shaped,with most cells adhering to the wall and a few cells floating.(4)Hoechst fluorescence results showed that the Control group showed weak blue light,and the model group showed strong blue fluorescence.The blue fluorescence intensity of PUE administration group was significantly decreased compared with the model group,and the blue light intensity of Edaravone group was weak and close to the Control group.(5)AV-PI flow cytometry results showed that the apoptosis rate of model group was41.5%,PUE significantly decreased the apoptosis rate to 19.2%,and Edaravone group decreased the apoptosis rate to 11.4%.(6)Flow cytometry results showed that compared with OGD/R group,the content of ROS in HT22 cells in PUE group was decreased,and the content of ROS in PI3 K inhibitor LY294002 group was significantly increased.(7)Compared with the Control group,the activity levels of ROS and MDA in OGD/R group increased,while the activity levels of SOD,CAT,GSH and GSH-px decreased;Compared with OGD/R group,the activity levels of ROS and MDA in LY294002 group increased,while the activity levels of SOD,CAT,GSH and GSH-px decreased,which were reversed and tended to normal after PUE treatment.(8)Western blotting showed that compared with the Control group,the expressions of pPI3 k,p-Akt,Cytoplasm Nrf2 and Keap1 were decreased in the OGD/R group,while the expressions of Nucleus Nrf2 and ho-1 were increased.Compared with OGD/R group,the expression of Cytoplasm Nrf2 and Keap1 in LY294002 group increased,while the expression of p-PI3 K,p-Akt,Nucleus Nrf2 and HO-1 decreased.PUE treatment group can reverse the above situation.(9)The results of molecular docking confirmed that PUE could dock with PI3K/Akt structure.(10)DARTS and CETSA experiments confirmed that PUE could bind PI3 K.(11)The results of co-immunoprecipitation verified the coupling of Keap1 and Nrf2 protein,and PUE could promote the dissociation of Keap1 and Nrf2 and play a role in inhibiting oxidative stress.Conclusion:(1)PUE has a significant protective effect on the ischemic brain tissue and HT22 cells after OGD/R in MCAO/R rats,and this protective effect is related to the improvement of intracellular oxidative stress level in the ischemic area.PUE can activate PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress,thus effectively protecting the brain tissue of MCAO/R rats and HT22 cells after OGD/R. |
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