| Ischemia stroke.Is is a cerebral vascular disease caused by vascular embolism,which can cause permanent death of tissue cells in ischemic brain areas and bring sequelae such as hemiplegia.Because the time window of its treatment is very limited,the key to the treatment of ischemic stroke is to save the neurons that have not died in the ischemic penumbra in time.Recent studies have shown that ischemic stroke can also cause the damage and dysfunction of the endoplasmic reticulum,activate its downstream signal pathway,participate in the occurrence and development of ischemia-reperfusion injury,and have an important impact on the apoptosis or survival of nerve cells.Carthamus tinctorius L.,as a blood activating and stasis removing drug,has the effects of activating blood circulation and removing stasis,dredging meridians and relieving pain.Among them,HSYA is a representative water-soluble quinchalone C-glycoside pigment,which is also a quality marker of safflower.Previous studies have shown that HSYA can penetrate the blood-brain barrier and has a certain protective effect on brain microvascular endothelial cells damaged by OGD/R,and this neuroprotective effect may be related to reducing oxidative stress.Therefore,the purpose of this study was to investigate whether HSYA can treat ischemic stroke through GRP78/PERK/CHOP pathway in vitro and in vivo.ObjectiveTaking the GRP78/PERK/CHOP signal pathway as the entry point,this paper discusses whether the neuroprotective mechanism of HSYA on ischemic stroke is related to GRP78/PERK/CHOP signal pathway,provides a new idea for clarifying the neuroprotective mechanism of HSYA on ischemic stroke,and provides an experimental basis for the clinical promotion and application of HSYA.Method1.Effects of HSYA on MCAO/R rats through GRP78/PERK/CHOP signaling pathway(1)The MCAO/R model of rats was established by the modified thread embolization method,and the rats with successful modeling were evaluated by neurobehavioral score.(2)The volume of cerebral infarction was detected by TTC staining and analyzed by Image J.(3)Brain histopathology of rats in each group was detected by HE staining.(4)Tunel-Neu N staining was used to detect the apoptosis rate of hippocampal neurons in each group.(5)The ultrastructure of hippocampal neurons was observed by transmission electron microscopy.(6)Western Blotting was used to detect the expressions of pathway-related proteins GRP78,PERK,p-PERK,CHOP,Bcl-2 and Bax in hippocampal neuron tissues of rats in each group.2.Protective effect of HSYA on HT22 cells injured by OGD/R(1)Establishment of OGD/R model.(2)Screening of safe concentration and optimal intervention concentration of HSYA.(3)LDH lactate dehydrogenase cytotoxicity test.(4)Hoechst33258 staining was used to detect apoptosis.(5)Flow cytometry Annexin V-PI staining was used to detect the apoptosis rate(6)Western blotting was used to detect the protein expression of Bcl-2 and Bax in hippocampal neurons of each group.3.The protective effect of HSYA on HT22 cells injured by OGD/R through GRP78/PERK/CHOP signaling pathway(1)CCK-8 was used to detect the cell viability of each group.(2)LDH content in HT22 cells of each group was determined by lactate dehydrogenase method.(3)The morphological changes of endoplasmic reticulum were observed by transmission electron microscopy.(4)The fluorescence intensity of calcium ions was observed by flow cytometry.(5)Flow cytometry was used to detect the apoptosis rate in each group.(6)The expression levels of GRP78,CHOP,Bcl-2,Bax,PERK,P-PERK,cleaved-caspase3 and cleaved-caspase9 proteins were detected by Western Blotting.(7)PERK was transfected with oerna,and the binding expression of GRP78 and perk was detected by Western blotting.(8)The connection between GRP78 and PERK protein was detected by immunofluorescence double labeling.(9)The protein expression was detected by immunofluorescence.(10)The expressions of GRP78,PERK,e IF2α,ATF4 and CHOP were detected by Western blot.Results1.Effects of HSYA on MCAO/R rats through GRP78/PERK/CHOP signaling pathway(1)HSYA can effectively reduce neurobehavioral score and cerebral infarction volume in MCAO/R rats.(2)HSYA can significantly improve the brain histopathology of MCAO/R rats.(3)Tunel-Neu N staining showed that HSYA could significantly reduce the apoptosis rate of hippocampal neurons in MCAO/R rats.(4)TEM results showed that,compared with the sham operation group,the endoplasmic reticulum was severely dilated and the structure of the endoplasmic reticulum was disordered in the model group.After treatment with HSYA and GSK2606414 inhibitors,the above injuries were reduced and the endoplasmic reticulum dilation was improved.(5)Western Blotting results showed that when PERK was suppressed,HSYA could significantly down-regulate GRP78,p-PERK,PERK,CHOP,Bax and up-regulate the expression of Bcl-2 compared with the model group.1.Protective effect of HSYA on HT22 cells injured by OGD/R(1)CCK8 results show that HSYA is between 5-140μM in 20-80μM dose range μ At m,the effect was better and the cell viability increased significantly.Compared with control group,the cell viability of OGD/R group decreased to about 50%;HSYA significantly increased the activity of HT22 cells and improved cell damage after administration.(2)The results of LDH kit showed that the release of LDH in the supernatant of HT22 cells increased significantly 4 hours after OGD injury.The release of HSYA decreased after treatment.(3)Hoechst fluorescence results showed that the high-dose HSYA group showed weak blue light,and the model group showed strong blue fluorescence.The blue fluorescence intensity of the low and medium dose HSYA group was significantly weakened compared with the model group,and the NMDP blue light showed weak and close to the normal group.(4)Av-pi flow cytometry showed that the apoptosis rate of the model group was 51.5%,which was significantly different from that of the control group.Compared with OGD/R group,low,medium and high doses of HSYA significantly reduced the apoptosis rate to33.2%、26.1% 、16.7%and NMDP group significantly reduced the apoptosis rate to10.2%.(5)Western blotting results showed that when OGD/R was injured,HSYA and NMDP could significantly down regulate the expression of Bax and up regulate the expression of Bcl-2 compared with the model group.conclusionHSYA can protect HT22 cells from OGD/R injury.3.The protective effect of HSYA on HT22 cells injured by OGD/R through GRP78/PERK/CHOP signaling pathway(1)CCK8 results showed that after OGD/R injury,cell viability decreased significantly in the model group and after using TM agonists,which proved that endoplasmic reticulum stress occurred.HSYA combined with agonists significantly improved cell viability.When combined with inhibitors,HSYA also played a role in protecting cell viability,showing a significant difference compared with the model group.(2)LDH results showed that the release of LDH increased significantly in the model group and TM group,while the release of LDH was significantly reduced in the inhibitor 4-PBA group,and the release of LDH was reduced in the group combined with HSYA.(3)The results of transmission electron microscope showed that in the model group,vesicles with rough endoplasmic reticulum structure and some nucleoli were broken.After treatment with HSYA,the endoplasmic reticulum of hippocampal neurons was slightly damaged and the vesicles were arranged neatly.The phenomenon of apoptosis has been greatly improved.(4)Calcium ion results showed that compared with the model group,HSYA group,inhibitor combined with HSYA group and agonist combined with HSYA group significantly inhibited the release of calcium ions,and there was a significant difference compared with the model group.(5)Av-pi flow cytometry showed that the apoptosis rate of model group was 42.3%,which was significantly different from that of control group.The apoptotic rate of TM group was 52.4%,and that of HSYA combined group decreased to 31.3%.Compared with OGD / R group,HSYA significantly reduced the apoptotic rate to 21.9%,and the inhibitor combined with HSYA significantly reduced the apoptotic rate to 9.9%.(6)For the protein expression of GRP78,chop,Bax,PERK,p-PERK,cleaved-caspase3 and cleaved-caspase9 in HT22 cells,compared with the control group,the protein expression of GRP78,chop,Bax,PERK,p-PERK,cleaved-caspase3 and cleaved-caspase9 in the model group increased significantly.Compared with OGD / R group,HSYA group,TM + HSYA,4-PBA and 4-PBA + HSYA group could inhibit the protein levels of GRP78,chop,Bax,PERK,p-PERK,cleaved-caspase3 and cleaved-caspase9 in HT22 cells.Compared with the control group,the expression of Bcl-2 decreased significantly.Compared with the model group,the protein expression increased significantly.(7)PERK was transfected with oe RNA lentivirus,and increased PERK expression was detected by Western Blotting.(8)The immunofluorescence double labeling showed that under normal conditions,the two cells were combined,but the cells in OGD/R group were deactivated and dissociated.The combination of fluorescence expression of both cells became less,and the combination of the two types of cells in the overexpressed PERK group was less.After HSYA treatment,GRP78 and PERK gradually moved from the dissociated state to the binding state,and the fluorescence of the two cells was increased.(9)Immunofluorescence showed that CHOP expression in OGD/R and Oe-PERK groups was significantly higher than that in Control and Oe-NC groups.CHOP protein expression in the cytoplasm decreased significantly after HSYA treatment.(10)Western blot results showed that the protein expressions of GRP78,PERK,e IF2α,ATF4 and CHOP were increased in OGD/R group and Oe-PERK group,which were significantly different from those in HSYA and Oe-PERK+HSYA group.conclusionHSYA can inhibit apoptosis mediated by GRP78/PERK/CHOP signaling pathway in OGD/R-injured HT22 cells,and has a protective effect on ischemia-reperfusion injury of nerve cells. |
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