The Therapeutic Mechanisms Of Gualou-Guizhi Decoction On The Rats With Cerebral Ischemia-Reperfusion Injury Via ERS-associated PERK/ATF4 Signaling Pathway

Objective:The MCAO model made by suture was established to explore the neuroprotective effects of Gualou-Guizhi Decoction(GLGZD)on rats with cerebral ischemia-reperfusion injury,and to further study whether GLGZD exerts its neuroprotection to promote the survival of the nerve cells by improving the endoplasmic reticulum stress(ERS)via regulating the Protein kinase R(PKR)-like ER kinase/Activating transcription factor 4(PERK/ATF4)signaling pathway,which provides the experimental basis for the clinical and scientific research on the rehabilitation of nerve functions after stroke.Method:(1)Model establishment,grouping and administration:The MCAO model was established by the classical suture method.After wide awake,all the rats were measured with longa neurological score.The score during 2 to 4 was regarded that the model has been established,then the rats can be utilized in the follow experiments.Next,those rats were randomly divided into 5 groups,including MCAO group,GLGZD low-dose group(GLGZD L),GLGZD middle-dose group(GLGZD M),GLGZD high-dose group(GLGZD H)and NMDP group.In addition,the Sham group was set as the blank control.All the rats were administrated once a day for 7 d.(2)The therapeutic effects of GLGZD on MCAO rats:After 7 d’ regulation,we used longa neurological score to evaluate the behavioral dysfunction of rats caused by neurological impairment.The volume of cerebral infarction was detected by TTC staining.The pathological change of nerve cells in ischemic cortex was contained by HE staining.Furthermore,the Tunel method was employed to acquire the apoptosis rate of ischemic neurons in cortex(3)The influence of GLGZD on the ERS-associated factor expression:We used the specific fluorescent probe to detect the concentration of Ca2+and ROS.The mRNA expression of GRP78,ATF4,CHOP,Ero-1α,Bcl-2,IP3R,SERCA and Bax in ischemic cortex were detected by qRT-PCR.The total protein expression of GRP78,PERK,p-PERK,eIF2α,p-elF2α,ATF4,CHOP,Bcl-2,Caspas-3,cleaved Caspase-3,Ero-1α,SERCA 2α,Bax and nucleoprotein for ATF4 were measured by Western-Blot.What’s more,the immumofluorescence and immunohistochemistry methods were conducted to detect the protein expression of GRP78 and IP3R respectively.In this section,we hope to use the methods mentioned above to explore the possible mechanisms for the neuroprotection of GLGZD.Results:1.The therapeutic effects of GLGZD on MCAO rats:Compared with MCAO group,the neurological score,infarct volume and cerebral cortex neurons apoptosis rate of rats were significantly reduced after GLGZD administration by(P<0.05,P<0.01).In HE staining,the phenomenon of apoptosis and necrosis such as cell arrangement disorder,cell structure destruction,cytoplasm reduction and agglutination,nucleus contraction and deep staining were performed in the ischemic cortex of MCAO group rats,which accompanied by interstitial edema and inflammatory infiltration.After GLGZD treatment,the apoptosis and necrosis phenomenon were quite improved.2.The influence of GLGZD on the ERS-associated factors expression in PERK/ATF4 pathway:(1)In terms of the Ca2+ and ROS concentration,the Ca2+ and ROS concentration of ischemic cortex in GLGZD groups were decreased compared with MCAO groups.This results are of significant statistically different(P<0.05,P<0.01).(2)The results conveyed from qRT-PCR detection reflected that the mRNA expression of GRP78,ATF4,CHOP,IP3R,Caspase-3,Ero-lα,Bax were significantly down-regulated(P<0.05,P<0.01)and SERCA2a as well as Bcl-2 were obviously up-regulated(P<0.05,P<0.01)in GLGZD groups compared with MCAO group.(3)The results of ER-associated protein expression detected by Western-Blot were showed as follow.① After modeling,the total protein expression of GRP78,ATF4,CHOP,Ero-la,Bax and the nucleoprotein of ATF4 were up-regulated(P<0.01)in ischemic cortex,while the SERCA2a and Bcl-2 were significantly down-regulated(P<0.01).In GLGZD groups,the total protein expression level of GRP78,ATF4,CHOP,Ero-1α,Bax and the nucleoprotein of ATF4 were lower than MCAO group(P<0.05,P<0.01),while the SERCA2αand Bcl-2 expression were much higher than former(P<0.05,P<0.01).② In the ischemic cortex of MCAO group rats,the ischemia-reperfusion injury promoted the phosphorylation of PREK and eIF2α,as well as activated Caspase-3(P<0.05,P<0.01).After being administrated by GLGZD,the activities of p-PERK,p-eIF2a and cleaved Caspase-3 were obviously inhibited(P<0.05,P<0.01).(4)The protein expression of GRP78 was detected by immunofluorescence.The result showed that the GRP78 was significantly up-regulated in the ischemic cortex of rats after modeling(P<0.01),and the protein was located in cytoplasm GLGZD could effectively reduce the expression level of GRP78(P<0.05,P<0.01).(5)The protein expression of IP3R was detected by immunohistochemistry.The result showed that IP3R was main located in nucleus and cytoplasm.what’s more,the protein expression of IP3R in the ischemic cortex of rats was increased significantly(P<0.01),and GLGZD could effectively reduce the expression level of IP3R(P<0.01).Conclusion:Generally,the results mentioned above indicated that GLGZD exert neuroprotective effects by inhibiting the apoptosis of nerve cells in ischemic cortex,improving the pathology state of the cortical neurons in ischemic area and reducing the cerebral infarction volume.Furthermore,its mechanisms have close relation with the inhabitation of ERS via the PERK/ATF4 signaling pathway.