Rehmannia Glutinosa Yinzi Regulates The Mechanism Of Endoplasmic Reticulum Stress In AD Mice With Energy Generation Disorder By PERK/eIF2α

Background:Alzheimer’s disease(AD)is a neurodegenerative disease characterized by cognitive and memory impairment.With the increasing trend of population aging in China,AD has become the sixth killer after cancer,hypertension and other diseases.Energy metabolism disorder plays a key role in the early initiation and evolution of AD.Endoplasmic reticulum stress caused by energy metabolism disorder is the core pathological change of AD.Dysfunction of energy metabolism can lead to disorder of endoplasmic reticulum homeostasis,aggregation of unfolded or misfolded proteins and hindered protein processing,which can activate the unfolded protein response to UPR.After phosphorylation of PERK/eIF2alpha,the downstream signaling pathway of UPR can increase the deposition of Abeta and aggravate AD-related neurodegeneration by increasing the level of BACE1.On the other hand,after phosphorylation of PERK and eIF2alpha,the expression of ATF4 and transcription factor CHOP were up-regulated in turn.After activation of CHOP,the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were further up-regulated to induce neuronal apoptosis,leading to synaptic loss,which resulted in neurological damage and cognitive decline.These results suggest that the inhibition of ERS-induced Abeta deposition and neuronal apoptosis by regulating PERK/eIF2alpha pathway and the improvement of energy metabolism disorder may be an important way to slow down AD-related neurodegeneration.The onset of AD is insidious,the progress of pathology is slow and irreversible.At present,most of the treatment methods mainly focus on intervention after onset,which can only improve and alleviate symptoms to a certain extent,and the clinical effect is not ideal.On the basis of long-term clinical practice,traditional Chinese medicine has formed a basic understanding of AD.Kidney deficiency and phlegm obstruction are its basic pathogenesis.Benefiting the kidney and resolving phlegm are effective therapies for clinical treatment of AD.Objective:Dihuang Yinzi is a classical prescription for tonifying kidney and resolving phlegm.It has been proved that Dihuang Yinzi can play an anti-AD role in many ways.We believe that there may be some intrinsic relationship between the pathological process of apoptosis of neurons and the pathological process of phlegm blockade and myeloablation caused by deficiency of kidney essence in TCM theory.Therefore,whether Dihuang Yinzi can improve energy metabolism by regulating PERK/eIF2alpha pathway Endoplasmic reticulum stress caused by dysfunction,blocking the up-regulation of BACE1,inhibiting the accumulation of Abeta,inhibiting the activation of ATF4/CHOP pathway,regulating the abnormal expression of apoptosis-related proteins,slowing down neuronal apoptosis,and exerting the mechanism of anti-AD,tonifying kidney and eliminating phlegm are the main topics of this study.In order to improve the early energy metabolism disorder,protect the homeostasis of endoplasmic reticulum,inhibit the deposition of Abeta and neuron apoptosis,we used AD mice with energy disorder as the model to observe the effects of Dihuang Yinzi on learning and memory ability and the expression level of endoplasmic reticulum stress-related protein in AD mice with energy disorder.To explore the mechanism of Dihuang Yinzi in inhibiting endoplasmic reticulum stress induced by energy metabolism disorder by regulating PERK/eIF2alpha pathway.Materials and methods:1.AD mice model of energy metabolism disorder was established by intraperitoneal injection of 3-nitropropionic acid into 4-month-old APP/PS1 transgenic mice.They were randomly divided into blank control group,model group,positive drug(Anlishen)group,Dihuang Yinzi low,medium and high dose group.Except for the blank control mice,the other mice in each group were intraperitoneally injected with 100mg.kg-13-nitropropionic acid once.After the mouse model was established,the mice in each group were given medication.Continuous intragastric administration for 1 week.2.Morris water maze test was used to test the spatial learning and memory ability of mice.3.The contents of ATP,ADP and AMP in brain tissue of energy-impaired mice were detected by high performance liquid chromatography,and the energy charge(EC)was calculated.4.The expressions of GRP78,PERK,eIF2alpha phosphorylation,BACE1,ATF4,CHOP,Bcl-2 and Bax were detected by Western blot.5.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the expression of BACEl1gene.6.Enzyme-linked immunosorbent assay(ELISA)was used to detect the deposition of A beta in the brain of mice.To evaluate the effect of Dihuang Yinzi on brain A beta in AD mice with energy metabolism disorder by inhibiting PERK/elF2alpha pathway.Result:1.Morris water maze resultsIn the positioning navigation experiment,the escape latency of AD mice in the model group was significantly longer than that in the blank control group,indicating that the learning and memory abilities of AD mice with energy metabolism disorder were decreased;compared with the model group,the escape latency of AD mice with energy metabolism disorder was significantly shortened in each dose group of Dihuang Yinzi(P<0.05,P<0.01);in the space exploration experiment,the escape latency of AD mice with energy metabolism disorder was significantly shortened in each dose group of Dihuang Yinzi compared with the model group.To significantly increase the number of times of mice crossing the target platform area(P<0.01,P<0.01,P<0.01)and the residence time in the target quadrant(P<0.01,P<0.01),and reduce the residence time in the relative quadrant(P<0.05,P<0.01,P<0.01),it shows that Dihuang Yinzi can improve the spatial learning and memory ability of AD mice.2.Results of High Performance Liquid Chromatography(HPLC)Compared with the blank control group,ADP,AT P and EC in the brain tissue of model group mice were significantly decreased,and AMP content was significantly increased(P<0.01),indicating that 3-NP caused significant energy metabolism disorder in the brain tissue of AD mice.Compared with model group,ADP,AT P and EC in brain of mice in low,middle and high dosage groups of Dihuang Yinzi increased significantly,while AMP decreased significantly(P<0.05,P<0.01).The content of AMP,ADP,ATP and EC in the brain of model mice were affected by Arizon,but there was no statistical significance.3.Western blot results(1)Compared with the blank control group,the energy metabolism disorder induced by 3-NP could significantly increase the expression level of GRP78,PERK,eIF2alpha phosphorylation and BACE1 protein(P<0.01).Compared with the model group,the low,medium and high dose groups of Dihuang Yinzi could significantly reduce the expression level of GRP78(P<0.05,P<0.01,P<0.01),the expression level of PERK and eIF2alpha phosphorylation(P<0.01,P<0.01),and inhibit the expression level of BACE1protein(P<0.01,P<0.01,P<0.01).The positive drug group had no significant effect on the phosphorylation of PERK/eIF2alpha.The results showed that Dihuang Yinzi could inhibit the protein translation level of BACE1 by regulating the phosphorylation level of PERK/eIF2alpha pathway,thus alleviating the deposition of A beta.(2)Compared with the blank control group,energy metabolism disorder induced by 3-NP could significantly increase the expression of ATF4 and CHOP(P<0.01),down-regulate the expression of anti-apoptotic protein Bcl-2(P<0.01),up-regulate the expression of pro-apoptotic protein Bax(P<0.01),and induce neuronal apoptosis in brain tissue.Compared with the model group,the low,medium and high dose groups of Dihuang Yinzi could significantly reduce the expression of ATF4 and CHOP protein(P<0.05,P<0.01,P<0.01),up-regulate the expression of anti-apoptotic protein Bcl-2(P<0.05,P<0.01,P<0.01),down-regulate the expression of apoptotic protein Bax(P<0.05,P<0.01,P<0.01),and slow down the apoptotic damage of neurons in brain tissue.4.Real-time PCR resultsCompared with the blank control group,the level of BACE1mRNA in the model group decreased,but there was no statistical significance.The results showed that the energy metabolism disorder induced by3-NP might inhibit the transcription of BACE1,but there was no statistical significance.Rehmannia Decoction and Lishen had no significant effect on the level of BACE1 mRNA,indicating that Rehmannia Decoction and positive drug groups had no significant effect on the transcription of BACE1 mRNA.5.ELISA resultsCompared with the blank control group,the content of Abeta 1-40 and Abeta 1-42 in the brain tissue of the model group mice increased significantly,indicating that the energy metabolism disorder induced by3-NP in AD mice increased the content of Abeta,resulting in the accumulation of insoluble oligomers and soluble oligomers.Compared with the model group,the low,medium and high dose groups of Dihuang Yinzi could significantly reduce the contents of Abeta 1-40 and Abeta 1-42 in the brain of model mice(P<0.01,P<0.01,P<0.01).The results showed that Dihuang Yinzi could inhibit the excessive accumulation of Abeta in the brain tissue of model AD mice.Conclusion:1.Rehmannia decoction can significantly improve the early energy metabolism disorder of AD,improve the learning and memory ability of AD mice with energy disorder,and alleviate cognitive impairment.2.Dihuang Yinzi can significantly improve the energy metabolism of brain tissue in AD mice with energy disorder.3.Rehmannia decoction can inhibit the activation of endoplasmic reticulum stress PERK/eIF2alpha signaling pathway and the translation of BACEl by inhibiting energy metabolism disorder,thereby reducing the content of Abeta in brain of AD mice with energy disorder,reducing the damage to brain tissue and delaying the course of AD;inhibiting the activation of ATF4/CHOP signaling pathway,then regulating the abnormal expression of apoptosis-related proteins Bcl-2and Bax,slowing the apoptosis of neurons and improving the course of AD.Brain tissue damage.