| Objective In this study,TX mice and human umbilical vein endothelial cells(HUVECs)were used to study the protective mechanism of Gandouling on Wilson's disease(WD)cerebrovascular damage in vivo and in vitro.To elucidate the mechanism of cerebrovascular damage in WD was related to the apoptosis mediated by endoplasmic reticulum stress PERK/e IF2?/CHOP signaling pathway.And to elucidate Gandouling,a traditional Chinese medicine,can inhibit apoptosis by regulating the endoplasmic reticulum PERK/e IF2alpha/CHOP signaling pathway,thus playing a protective role in WD cerebrovascular damage.By studied the molecular mechanism of Gandouling in protecting WD cerebrovascular damage,we could provide theoretical basis and scientific basis for clinical application of Gandouling,and provide new ideas for prevention and treatment of WD cerebrovascular damage by traditional Chinese medicine.The protection of Gandouling on cerebral vessels of TX mice induced by endoplasmic reticulum stress PERK/e IF2?/CHOP signaling pathway was observed in vivo and on HUVECs after copper stress was further verified by in vitro experiments of HUVECs simulating cerebral vascular endothelial cells,to further explore the mechanism of Gandouling on cerebral vascular protection in TX mice model with WD.Methods In vivo experiments--The protective mechanism of Gandouling on cerebrovascular in TX mice: 48 TX mice were randomly divided into model group,Gandouling group,Penicillamine group,Gandouling plus Penicillamine group,and 12 DL mice as control group.To observe the changes of v WF,TM and ACA in mice serum and the pathological changes of cerebrovascular in mice of each group,and then immunohistochemistry was used to observe the expression of VCAM-1,ICAM-1 and GRP78 in murine vascular endothelial cells.TUNEL was used to observe the apoptosis of cerebrovascular endothelial cells and Western Blot was used to observe the expression of PERK signaling pathway key protein in endoplasmic reticulum stress about brain tissues of mice in each group.In vitro experiments--The protective mechanism of Gandouling on copper-loaded HUVECs: The experiment was divided into six groups: normal group,control group,Gandouling low-dose group,Gandouling medium-dose group,Gandouling high-dose group and Salubrinal group.Flow cytometry was used to observe the effect of Gandouling on the apoptosis of HUVECs induced by copper stress,RT-q PCR was used to observe the effect of Gandouling on the expression of PERK,p-e IF2? and CHOP in HUVECs induced by copper stress,Western Blot method was used to observe the effect of Gandouling on the expression of PERK,p-e IF2? and CHOP in HUVECs induced by copper stress,the effect of Gandouling on caspase-3 activity in HUVECs induced by copper loading was observed by colorimetry.Results In vivo experiments--The protective mechanism of Gandouling on cerebral vessels in TX mice:(1)Serum vascular injury factors: The values of v WF,TM and ACA in the model group were significantly higher than those in the control group(P<0.01).Compared with model group,the values of v WF,TM and ACA in Gandouling group decreased,and the difference was significant(P<0.05),and the values of v WF,TM and ACA in Penicillamine group decreased,the difference was significant(P<0.05),the values of v WF,TM and ACA in Gandouling plus Penicillamine group decreased significantly(P<0.01).(2)Histopathological changes of cerebrovascular tissue: There was edema and degeneration of cerebrovascular endothelial cells in model group,but no edema and degeneration of cerebrovascular endothelial cells in control group,and the results showed that cerebrovascular injury occurred in model group.Compared with the control group,the pathological changes in GDL group and Penicillin group showed mild edema and degeneration of cerebrovascular endothelial cells.The results showed that Gandouling and Penicillamine could improve cerebrovascular injury.Gandouling and Penicillamine combined treatment had the best protective effect on cerebrovascular injury in WD mice.(3)The expression of endothelial cell injury factors VCAM-1,ICAM-1 and GRP78 was observed by immunohistochemistry: In control group,VCAM-1,ICAM-1 and GRP78 were not positive stained in brain slices of DL mice.Compared with the control group,TX mice in the model group showed obvious VCAM-1,ICAM-1 and GRP78 positive staining of blood vessels,which were brown and yellow under the microscope,and there were significant statistical differences(P<0.01).Compared with model group,the positive staining of VCAM-1,ICAM-1 and GRP78 in Gandouling group or Penicillamine group was significantly less(P<0.05),the positive staining of VCAM-1,ICAM-1 and GRP78 in Gandouling plus Penicillamine group was significantly lower than that in model group with significant statistical difference(P<0.01).(4)Observation of endothelial cell apoptosis by TUNEL: There was no obvious fluorescence labeling on cerebral vascular endothelial cells of DL mice in the control group.Compared with the control group,the model group showed obvious fluorescence labeling,and the AOD ratio has significant statistical difference(P<0.01).Compared with the model group, the fluorescence labeling of mouse cerebrovascular endothelial cells in Gandouling group or Penicillin group was weakened,and the AOD ratio has statistical difference(P<0.05),the fluorescence labeling of mouse cerebrovascular endothelial cells in Gandouling plus Penicillamine group was significantly weakened with significant statistical differences(P<0.01).(5)Western Blot method to observe the expression of key proteins in PERK signaling pathway: There was no significant protein expression of PERK,p-e IF2?,CHOP,caspase-12 and Caspase-3 in the cerebrovascular tissues of DL mice in the control group.Compared with the control group,the protein expression of PERK,p-e IF2?,CHOP,caspase-12 and Caspase-3 in the model group was significant increased,the difference had significant statistical(P<0.01).Compared with the model group,the protein expression of PERK,p-e IF2?,CHOP,caspase-12 and Caspase-3 in Gandouling group or Penicillin group decreased,and the difference had statistical(P<0.05),the protein expressions of PERK,p-e IF2?,CHOP,caspase-12 and Caspase-3 in Gandouling plus Penicillamine group were obvious decreased,and the difference had significant statistical(P<0.01).In vitro experiments--The Protective mechanism of Gandouling on copper-loaded HUVECs:(1)Flow cytometry was used to observe the apoptosis of HUVECs: Compared with the normal group,the control group cultured HUVECs under simulated copper loading for 24 hours significantly increased the apoptotic rate of HUVECs(P<0.01).Compared with the control group,Gandouling inhibits HUVECs apoptosis in a concentration-dependent manner in low,medium and high dose groups(P < 0.05,P < 0.01),Salubrinal(30 mol/l),a specific e IF2? phosphatase inhibitor,could significantly inhibit HUVECs apoptosis(P < 0.01).(2)RT-q PCR to observe the expression of PERK,p-e IF2? and CHOP m RNA:Compared with the normal group,the control group cultured HUVECs under simulated copper stress for 24 hours significantly increased the expression of PERK,p-e IF2?,CHOP m RNA in HUVECs(P<0.01).Compared with the control group,the expression of PERK,p-e IF2? and CHOP m RNA in HUVECs was inhibited in a concentration-dependent manner by Gandouling at low,medium and high doses(P<0.05,P<0.01),Salubrinal(30mol/l),a specific e IF2? phosphatase inhibitor,could significantly inhibit the expression of p-e IF2? and CHOP m RNA in HUVECs(P<0.01),but it did not interfere with the expression of PERK m RNA(P>0.05).(3)Western Blot method to observe the expression of PERK,p-e IF2? and CHOP protein: Compared with the normal group,the protein expression of PERK,p-e IF2? and CHOP in HUVECs cultured for 24 hours under simulated copper stress in the control group was significantly increased(P<0.01).Compared with the control group,the protein expression of PERK,p-e IF2?and CHOP in HUVECs was inhibited in a concentration-dependent manner by Gandouling at low,medium and high doses(P<0.05,P<0.01),Salubrinal(30mol/l),a specific e IF2? phosphatase inhibitor,could significantly inhibit the protein expression of p-e IF2? and CHOP in HUVECs(P < 0.01),but had no effect on the PERK(P > 0.05).(4)Observation of caspase-3 activity by colorimetry: Compared with the normal group,the activity of Caspase-3 of HUVECs was significantly increased in the control group after 24 hours of culture under simulated copper stress(P < 0.01).Compared with the control group,the activity of Caspase-3 in HUVECs was inhibited in a concentration-dependent manner by Gandouling at low,medium and high doses(P<0.05,P<0.01),Salubrinal(30mol/l),a specific e IF2? phosphatase inhibitor,could significantly inhibit the activity of Caspase-3 in HUVECs(P < 0.01).Conclusions In vivo experiments:(1)Cerebrovascular injury and pathological changes of vascular endothelial cells were observed in TX mice with Wilson's disease,under light microscopy,endothelial cells degenerate and necrosis in varying degrees,ultrastructure changes and endothelial cells swell.At the same time,the levels of vascular injury factors(v WF,TM,ACA)in serum are increased.Immunohistochemistry showed that the expression of VCAM-1 and ICAM-1 in vascular endothelial cells increased.(2)The TX mice with Wilson's disease,cerebral vessels were damaged,vascular endothelial cells were apoptotic,and the expression of GRP78,a key protein of endoplasmic reticulum stress signaling pathway was activated.(3)The apoptosis of cerebrovascular endothelial cells in TX mice with Wilson's disease was significantly increased and the expressions of PERK,p-e IF2?,CHOP,caspase-12 and caspase-3 protein in endothelial cells were significantly increased.Therefore,it was inferred that the apoptosis of cerebrovascular endothelial cells was through the apoptotic signal pathway of PERK/e IF2?/CHOP.(4)Gandouling can improve cerebral vascular injury in TX mice,alleviate endothelial cell swelling,reduce the level of serum vascular injury factors(v WF,TM,ACA),reduce the expression of vascular injury factors(VCAM-1,ICAM-1)in vascular endothelial cells,reduce the apoptosis and the expression of PERK,p-e IF2?,CHOP,caspase-12,caspase-3 protein in vascular endothelial cells.Therefore,we speculated that Gandouling had protective effect on the cerebrovascular of TX mice by interfering with the endoplasmic reticulum stress PERK/e IF2?/CHOP apoptotic signaling pathway.In vitro experiments:(1)Copper loading can cause damage and apoptosis of HUVECs,and can reduce the apoptotic rate of HUVECs.Full concentration of Gandouling had better therapeutic effect and more obvious protective effect on HUVECs.(2)Copper stress induced apoptotic rate of HUVECs was significantly increased,the m RNA and protein expression of PERK,p-e IF2? and CHOP,and the caspase-3 activity in HUVECs were significantly increased.So it was concluded that the apoptosis of HUVECs was mediated by PERK/e IF2?/CHOP endoplasmic reticulum stress signaling pathway.(3)Gandouling can improve copper-induced injury in HUVECs,reduce the m RNA and protein expression of PERK,p-e IF2? and CHOP in HUVECs and reduce the activity of caspase-3.Therefore,it was speculated that Gandouling can inhibit cell apoptosis through PERK/e IF2?/CHOP endoplasmic reticulum stress signaling pathway,thus playing a protective role on blood vessels of brain... |
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