Explore Buyang Huanwu Decoction Inhibiting Endoplasmic Reticulum Stress-Mediated Apoptosis And Reducing Cerebral Ischemia-Reperfusion Injury Based On Perk Pathway

Cerebral ischemia-reperfusion（I/R）injury is a common pathophysiological change during reperfusion recovery.It is affected by many factors such as time and intensity of reperfusion,and has a relationship with many mechanisms for inflammatory response,autophagy and apoptosis.Recent studies have shown that endoplasmic reticulum stress-mediated apoptosis plays a vital role in the occurrence and development of cerebral I/R injury,and determines the final infarct volume of brain tissue.Glucose regulated protein 78 k D（GRP78）is a marker protein of ER stress,and the PERK pathway is a vital signaling pathway that mediates ER stress-dependent apoptosis.Detection of Bcl-2 family related proteins can also respond to endoplasmic reticulum stress（ERS）-mediated apoptosis.Ischemic stroke belongs to the category of"stroke"in traditional Chinese medicine.Qi deficiency and blood stasis is the main pathogenesis of this disease.Buyang Huanwu decoction（BYHWD）is a classic prescription for the treatment of Qi deficiency and blood stasis syndrome in apoplexy in traditional medicine and has achieved positive clinical effects.Previous studies in our laboratory have shown that BYHWD can decrease cerebral I/R injury in rats,but whether ERS-mediated apoptosis is involved in that has not been reported.This study using SD rat middle cerebral artery occlusion（MCAO）model to simulate the cerebral I/R injury,by observing the behavior of rats to change,the neurons in the pathological morphology,ultrastructure of endoplasmic reticulum stress change,and ERS iconic protein and apoptosis related proteins expression to explore the BYHWD regulation of ERS inhibits apoptosis effects on cerebral I/R injury.Otherwise,using PC12 cells of oxygen lack of sugar/after oxygen after sugar（OGD/R）model to simulate the cerebral I/R injury,and to give PERK inhibitor intervention,through the CCK-8 test cell activity,immunohistochemical method to detect PERK protein involved in signaling pathways to explore BYHWD inhibiting endoplasmic reticulum stress-mediated apoptosis and reducing cerebral ischemia-reperfusion injury based on PERK pathwayThe first part:Effects of Buyang Huanwu Decoction on nerve injury,expression of endoplasmic reticulum stress protein GRP78 and apoptosis protein in rats with cerebral ischemia reperfusionObjective:To explore the effects of BYHWD on nerve injury,ERS protein GRP78and apoptosis protein expression in rats with cerebral I/R.Methods:1.The male SD rats（120）were divided into the sham and the operated group randomly.The operated rats were established the MCAO model to simulate cerebral I/R injury.After cerebral ischemia for 2 h,reperfusion was respectively performed for 24 h（50 rats）and 72 h（50 rats）.The successful model rats were divided into model,BYHWD and Nimodipine group randomly.The BYHWD group rats were given BYHWD by gavage(14.3g·kg^-1)after recovery twice a day.Once after waking up,the Nimodipine group rats were given by intraperitoneal injection(10mg·kg^-1).2.Using Zea Longa score observes the neurological deficits.Using TTC staining to determine the cerebral infarction volume of rats.Toluidine blue method was used to observe the pathological changes of nerve cells at 24hours of reperfusion;hematoxylin-eosin（HE）method was used to observe the pathological changes of nerve cells at 72 hours of reperfusion.Observation of ER ultrastructural lesions at 72 hours after reperfusion by transmission electron microscope.Western blotting detects the expression difference of ERS marker protein GRP78 at different time points and the expression of apoptosis-related proteins Bax/Bcl-2 and Caspase-3 at 72 h after reperfusion.the GRP78 protein expression of rats’right cerebral cortex.3.Statistical MethodAll data were analyzed by Graph Pad Prism 8.0.2 software and presented as the mean±SD.The differences between groups of all experimental data were used by One-way analysis of variance（ANOVA）.P<0.05 was considered statistically significant.Results:1.Model success rate:A total of 100 rats were included in the operation group by random sampling,and models were screened by Zea Longa scoring method,among which 82 rats were scored 1-3,and the success rate of the model was 82%.2.The score of nerve function deficit showed that:the score of sham operation group was 0,which means no nerve function deficit.Compared with the sham,the scores of model group were increased,and the neurological function deficit was serious（P<0.05）.Compared with the model,the score of BYHWD group and Nimodipine group was decreased（P<0.05）.3.TTC staining showed that no cerebral infarction was observed in the sham operation group,but obvious infarction was observed in the model group（P<0.05）.Compared with model group,the volume in BYHWD group and Nimodipine group decreased（P<0.05）.4.Toluidine blue staining showed that the nerve cells in the sham operation group were regular and abundant,compared with sham group,the model group were deformed and necrotic,and the number of neurons was significantly reduced（P<0.05）,compared with the model group,BYHWD group neurons morphology tended to be regular,with more（P<0.05）.5.HE staining results:the cytoplasm and the background are pink in varying degrees,the nucleus is light blue,and the nucleolus is a dark blue spot.If the chromatin condenses,dark blue spots can be seen.The sham group showed that the morphology of neurons was regular,the nucleus was nearly round and light blue,the nuclear membrane was smooth and intact,and a dark blue nucleolus was seen in the middle.In some cells,chromatin condensed and deeply stained in the nucleus dark blue patches.Compared with the sham,neurons in the model group were deformed,with irregular nuclei,fewer light blue nuclei,ruptured nuclear membrane,no or rare nucleoli,and dark blue patches of chromatin coagulation.Compared with the model,the BYHWD group’s cell condition was improved,the morphology tended to be regular,the nucleus was nearly round,the nuclear membrane was smooth and complete,nucleoli were seen in the middle,and some cells had dark blue patches of chromatin condensation phenomenon.6.The ultrastructure of transmission electron microscope showed that the rough ER with lots of ribosomes attached was regular in the sham group.Compared with the sham,the rough ER in the model is severely dilated and is accompanied by ribosome shedding.The BYHWD and the Nimodipine group had less rough ER expansion and more abundant ribosomes to compare with the model.7.Western Blotting detection of ERS protein GRP78 at 24 h and 72 h after reperfusion showed that the GRP78’expression was low in the sham group at different times.Compared with the sham,the protein expression in model group was increased（P<0.05）.Compared with the model,the GRP78protein in BYHWD group was increased at 24 h after reperfusion（P<0.05）,and decreased at 72 h after reperfusion（P<0.05）.Nimodipine group and BYHWD showed the same trend.8.Western Blot analysis of apoptosis-related proteins Bax/Bcl-2 and Caspase-3 at 72 h after reperfusion showed that the protein expression of Bax/Bcl-2 and Caspase-3 in model group was increased compared with sham group（P<0.05）.Compared with the model,the expression level of BYHWD and Nimodipine group was decreased（P<0.05）.Summary:BYHWD can improve the neurological function of MCAO model rats,reduce cerebral infarction volume,improve cell morphology,and reduce cell ultrastructure changes.At the same time,BYHWD can increase the expression of GRP78 at 24 h of ischemia-reperfusion,inhibit the expression of GRP78 at 72 h of ischemia-reperfusion,and reduce the expression of apoptosis-related proteins Bax/bcl-2 and Caspase-3.It is speculated that BYHWD may reduce cerebral I/R injury by inhibiting ERS-mediatedt apoptosis.The Second Part:Study on the mechanism of Buyang Huanwu Decoction in inhibiting endoplasmic reticulum stress-mediated cell apoptosis based on PERK pathway and alleviating hypoxia and hypoglycemia/reoxygenation and hypoglycemia injury in PC12 cellsObjective:To investigate the effect of BYHWD on OGD/R injury in PC12 cells by inhibiting ERS-mediated cell apoptosis based on PERK signaling pathway.Methods:1.PC12 cells were randomly divided into blank group,BYHWD with different concentrations of drug serum group and negative control with different concentrations of serum group.The growth status of cells in each group was observed under a microscope.After the cell density reached 90%,the cell activity of each group was detected by CCK-8,and the effect of the serum containing BYHWD on the activity of PC12 cells was observed.2.PC12 cells were randomly divided into blank,model,BYHWD with different concentrations of drug-containing serum group and negative control with different concentrations of serum group.Except the blank group,the other three groups were modeled with OGD for 2 h and reoxygenation and glucose reoxygenation for 24 h to simulate cerebral I/R injury.The effects of drug serum of BYHWD on OGD/R injury were observed and the effective concentration was screened out.3.PC12 cells were randomly divided into blank,model,PERK inhibitor,BYHWD and negative control group.Except for blank group,the other 4groups were subjected to OGD for 2 h and reoxygenation and glucose reoxygenation for 24 h.The expression of PERK signaling pathway related protein p-e IF2αwas detected by immunohistochemistry.4.Statistical MethodAll data were analyzed by Graph Pad Prism software and presented as the mean±SD.The differences between groups of all experimental data were used by One-way analysis of variance（ANOVA）.P<0.05 was considered statistically significant.Results:1.Microscopic observation results:To compare with the blank,the cells of the negative groups and the BYHWD groups with different concentrations were in good condition,regular in morphology and abundant in quantity,without significant difference.2.The effect of BYHWD containing serum on normal PC12 cell viability:The OD value detected by CCK-8 represents cell viability.Compared with the blank group,there was no significant difference in the OD value of the negative control group with different concentrations and the different concentrations of BYHWD（P>0.05）,indicating that the serum containing BYHWD had no effect on the activity of normal PC12 cells.3.The effect of BYHWD containing serum on OGD/R injury of PC12cells:OD value represents cell viability.The blank group’s OD was the highest,indicating the best cell activity in this group.To compare with the blank,the cell activity in the model was lower（P<0.05）.To compare with the model group,the cell activity of the BYHWD 1/500 concentration medicated serum group was increased（P<0.05）,and the cell activity of the other groups was not significantly increased.The activity of the negative group at different concentrations was not significantly higher than that of the model group,indicating that BYHWD-containing serum can effectively reduce PC12 cells OGD/R damage,and the effective concentration is 1/500.4.The effect of BYHWD on PERK pathway:the nucleus is blue,the positive expression of p-e IF2αis brown-yellow,and the dark blue plaques in the nucleus may be chromatin condensation（the pathological feature of apoptosis）.The staining results showed that a small amount of cells in the blank group showed brown-yellow precipitation,the positive cell expression rate was low,and there was no dark blue plaque chromatin condensation.To compare with the normal,the model group had brown-yellow precipitation in the cytoplasm of the cells.The expression rate of positive cells was higher（P<0.05）,and dark blue plaques with chromatin condensation were seen in the nucleus.To compare with the model,the number of cells with brown particles in the PERK inhibitor group decreased,and the rate of positive cells decreased（P<0.05）.To compare with the model,some cells in the BYHWD serum group showed brown particles,and the positive expression rate was reduced（P<0.05）.A few cells showed deep staining of nuclei and chromatin condensation.Apoptosis morphology was reduced.There was no significant difference in cell activity between the negative and the model group.Summary:The effective concentration of BYHWD-containing serum is 1/500,which can enhance the activity of OGD/R PC12 cells,reduce the positive expression of p-e IF2αcells,and reduce the phenomenon of chromatin aggregation that represents apoptosis,indicating that BYHWD-containing serum can alleviate PC12 cells OGD/R damage may be related to inhibiting the PERK pathway,down-regulating the expression of p-e IF2α,and reducing ERS-mediated apoptosis.Conclusion:1.BYHWD can improve the neurological function of MCAO model rats,reduce cerebral infarction volume,improve cell morphology,and reduce cell ultrastructure changes.At the same time,BYHWD can increase the expression of GRP78 at 24 h of ischemia-reperfusion,inhibit the expression of GRP78 at 72 h of ischemia-reperfusion,and reduce the expression of apoptosis-related proteins Bax/bcl-2 and Caspase-3.It is speculated that BYHWD may reduce cerebral I/R injury by inhibiting ERS-mediated apoptosis.2.The effective concentration of BYHWD-containing serum is 1/500,which can enhance the activity of OGD/R PC12 cells,reduce the positive expression of p-e IF2αcells,and reduce the phenomenon of chromatin aggregation that represents apoptosis,indicating that BYHWD-containing serum can alleviate PC12 cells OGD/R damage may be related to inhibiting the PERK pathway,down-regulating the expression of p-e IF2αand reducing ERS-mediated apoptosis.