| ObjectiveTo observe the effect of Huangdi Anxiao capsule(HDAX)on endoplasmic reticulum(ER)stress mediated apoptosis pathway in the animal model of diabetic cognitive dysfunction(DCD),the neuroprotective mechanism of HDAX in improving learning and memory ability were discussed.Methods1 Establish the rat model of DCD10 SD rats were fed basic feed without any treatment as normal group.60 SD rats were fed with high-fat diet.High-fat fed rats were injected with STZ at the end of the 4th week.Fasting blood glucose was detected after 72 hours,which was higher than 11.1mmol/L and continued to the next experiment.After one week of preadministration,Aβ25-35 was injected with bilateral hippocampal to aggravate cognitive deficit.Then Morris water maze test was conducted to explore the escape lantency and space exploration ability,total cholesterol(T-CHO),triglyceride(TG),glycated serum protein(GSP)level,as well as HE staining in hippocampus,and immunofluorescence of Aβ deposition in hippocampus.2 Pharmacodynamics of HDAX on DCD ratsAfter 28 days treatment of high-dose,middle-dose and low-dose of HDAX,then compared with model group(STZ+Aβ25-35).Morris water maze experiment was conducted to test the changes in escape latency and spatial exploration ability of rats in each group.Biochemical indicators detect the level of fasting blood glucose and lipid level in each group.HE staining and immunofluorescence were used to observe the hippocampal changes and Aβ deposition in each group.3 Effects of HDAX on ER stress mediated apoptosis pathway in DCD ratsWestern blot was used to detect the expression of 7 key protein of GRP78,CHOP,Bcl-2,Bax,caspase-12,caspase-9,and caspase-3 in hippocampal of ER stress mediated apoptosis pathway.RT-qPCR was used to detect the mRNA content of 7 key mRNA GRP78,CHOP,Bcl-2,Bax,procaspase-12,procaspase-9,and procaspase-3 in the ER stress mediated apoptosis pathway.To investigate the effect of HDAX in ER stress mediated apoptosis pathway in DCD rats.Results1 Establishment of DCD rats(1)Changes of fasting blood glucose(FBG):Compared with normal group,FBG in model group was significantly increased(P<0.01).(2)Morris water maze results:Compared with normal group,the escape latency time significantly increased and the times of crossing the platform of model rats were significantly decreased(P<0.05,P<0.01).(3)Changes of blood lipid:Compared with normal group,TG,T-CHO levels in model group were significantly increased(P<0.01).(4)Changes of GSP:Compared with normal group,GSP levels in the model group was significantly increased(P<0.01).(5)Changes of hippocampal neuron damage:The neurons and nuclei of the normal group were shape completely,arranged closely and dye uniformly.The morphology of neurons in model group were arranged disorder,fewer neurons number and more dead nerve cells with small volume,cell nucleus shrinkage and rupture.(6)Changes of hippocampal Aβ deposition:Compared with normal group,a large amount of Aβ deposition were found in hippocampal region in model group.2 Pharmacodynamics of HDA X improving DCD rats(1)Changes of FBG:After 28 days treatment,compared with model group,FBG level in high-dose and middle-dose group,Done group and DMBG group were all decreased(P<0.05,P<0.01).(2)Morris water maze results:Compared with model group,the escape latency decreased in high-dose and middle-dose group,Done group(P<0.05,P<0.01);the frequency of crossing the platform increased in middle-dose group,DMBG group and Done group(P<0.01).(3)Changes of blood lipid:Compared with model group,TG,T-CHO levels in high-dose,middle-dose and low-dose of HD AX group,DMBG group and Done group were significantly decreased(P<0.05,P<0.01).(4)Changes of GSP:Compared with model group,GSP levels in rats in high-dose,middle-dose and low-dose of HDAX group,DMBG group and Done groups were significantly decreased(P<0.01).(5)Changes of hippocampal neuron damage:The hippocampal nerve cells in the middle-dose group of HDAX were closely arranged and stained evenly,with occasional neuron injury.In the high-dose and low-dose of HDAX group,DMBG group and Done group,the arrangement of hippocampal nerve cells were disordered,with some neurons were damaged and fell off.(6)Changes of hippocampal Aβ deposition:Compared with model group,the deposition of Aβ in hippocampus of rats in HDAX group,DMBG group and Done group were significantly decreased.These results showed that rats with DCD induced by Aβdeposition could reduce by the treatment of HDAX,thus playing a role in brain protection.3 Mechanism of HDAX inhibiting ER stress mediated apoptosis pathway(1)Western Blot results:Compared with normal group,GRP78,CHOP,Bax,Caspase-12,Caspase-9,and Caspase-3 protein expressions were significantly increased in model group(P<0.01).The expression of Bcl-2 protein was significantly decreased(P<0.01).After treatment,protein expressions of GRP78,CHOP,Bax,Caspase-12,Caspase-9 and Caspase-3 were significantly decreased in middle-dose group,DMBG group and Done group compared with model group(P<0.05,P<0.01).The expression of Bcl-2 protein was significantly increased(P<0.05,P<0.01).(2)RT-qPCR results:Compared with normal group,the mRNA expression of GRP78,CHOP,Bax,procaspase-12,procaspae-9 and procaspase-3 in the model group were significantly increased(P<0.01),and the mRNA expression of Bcl-2 were significantly decreased(P<0.01).After treatment,mRNA levels of GRP78,CHOP,Bax,procaspase-12,procaspase-9 and procaspase-3 in middle-dose group,DMBG group and Done group were significantly decreased(P<0.05,P<0.01),and mRNA levels of Bcl-2 were significantly increased(P<0.01),compared with model group.Conclusion1.Intraperitoneal injection of STZ combined with hippocampal injection of Aβ25-35 can build the model of DCD.2.HDAX capsule can reduce blood glucose and lipid levels and improve the damage of nerve cells,reduce Aβ deposition in hippocampus of DCD rats.3.The mechanism of HDAX capsule of neuroprotective effect on rats with diabetic cognitive dysfunction may be related to the reduction of cell apoptosis by inhibiting endoplasmic reticulum stress mediated apoptosis pathway. |
PDF Full Text Request