The Exploring Mechanism Of Dengzhan Shengmai Capsules In Improving Cognitive Impairment Of Alzheimer’s Disease Based On Endoplasmic Reticulum Stress

ObjectiveAlzheimer disease(AD)is the most common type of dementia among the elderly,overburdening the society and family,there is still no drug that can slow the progression of AD.Recent study has shown that Dengzhan Shengmai capsules(DZSM)might play a role in cognitive dysfunction of AD,but the underlying mechanism is still unclear.Endoplasmic reticulum(ER)stress is involved in the pathological process of AD.Abnormal accumulation of Aβ proteins within the ER can cause abnormal levels of ER stress,leading to neurodegeneration.Recent studies have reported that the transcriptional regulator glutamine-rich protein 1(QRICH1),as the downstream of ER stress pathway,was a key effector of cell fate under stress conditions.The present study aims to explore the improvement effect of DZSM against cognitive impairment of AD via reducing Aβ plaque aggregation from in vitro and in vivo experiments,network pharmacology and molecular docking studies,which may be achieved by regulating the ER stress pathway.Methods1.Effects of DZSM on cognitive function of APP/PS1 transgenic miceAPP/PS1 transgenic mice were used and treated with different doses(40,200 mg/kg/d)of DZSM for eight weeks.The learning and memory abilities of mice were tested by Morris water maze test,new object recognition test and open field test.2.Effect of DZSM on neuronal function of APP/PS1 transgenic miceAPP/PS1 transgenic mice were used and treated with different doses(40,200 mg/kg/d)of DZSM for eight weeks.Aβ in the brain of the mice was detected by immunohistochemical staining and Thioflavin T staining.Nissl staining was used to detect the neuronal morphology.The superoxide dismutase(SOD),glutathione peroxidase(GSHPx)and malondialdehyde(MDA)assay kit were performed to assess oxidative stress level.The acetylcholine(ACh),choline acetyltransferase(ChAT)activity and acetylcholinesterase(AChE)assay kit were performed to assess cholinergic system.TUNEL staining and western blot were used to detect neuronal apoptosis.3.Effect of DZSM on endoplasmic reticulum stress of APP/PS1 transgenic miceAPP/PS1 transgenic mice were used and treated with different doses(40,200 mg/kg/d)of DZSM for eight weeks.The levels of ER stress signaling pathway were detected by western blot and immunofluorescence staining,including BiP,PDI,P-PERK/PERK,PeIF2α/eIF2α,ATF4,CHOP,QRICH1,P-IRE1α/IRE1α,XBP1s,and ATF6.The active components of DZSM were screened through TCMSP and ETCM online database,PubChem and SwissTargetPrediction platform,then AutoDockTools 1.5.7 software was used to calculate and analyze the active components that optimally combined with QRICH1.4.Effect of DZSM on Aβ1-42-induced HT22 cell injury and its mechanismThe mouse hippocampal neuron cell line HT22 cell was used and treated with different concentrations of Aβ1-42 and DZSM medicated serum.The cell viability was detected by CCK-8,and the levels of ER stress signaling pathway were detected by western blot,including BiP,PDI,P-PERK/PERK,P-eIF2α/eIF2α,ATF4,CHOP,QRICH1,PIRE1α/IRE1α,XBP1s,and ATF6.Results1.Effects of DZSM on the cognitive function of APP/PS1 transgenic miceOpen field test results showed that the central zone distance,residence time and crossing frequency of APP/PS1 mice were decreased,and increased significantly after treatment with DZSM.New object recognition test results showed that APP/PS1 mice rarely explored new objects,and their cognitive index was significantly reduced,after treatment with DZSM,the ability to distinguish familiar object and novel object increased,and the cognitive index was significantly increased.Morris water maze test results showed that a longer escape latency was observed in APP/PS1 group,and their locomotor trajectory was disordered.The time spend in target quadrant and crossing times of the target platform decreased significantly.After treatment with DZSM,the escape latency was significantly reduced,the movement trajectory was clear,the time spend in target quadrant and crossing times of the target platform was significantly increased.These results showed that DZSM could improve the learning and memory defects in APP/PS1 mice.2.Effect of DZSM on neuronal function of APP/PS1 transgenic miceIHC staining and Thioflavin T staining results showed that a large number of Aβ protein plaques gathered in cortex and hippocampus(CA1)of APP/PS1 mice,the aggregation of plaques was significantly down-regulated after treatment with DZSM.The results showed that DZSM could reduce the accumulation of Aβ plaques in the brain of APP/PS1 mice.Nissl staining results showed that Nissl’s bodies were few or missing,and the neurons exhibited degenerated or shrunken neuronal vacuoles,irregularly arranged,weakly stained both in the hippocampal(CA1)and cortical areas of APP/PS1 mice.After DZSM administration,deeper and denser Nissl bodies were found as well as neurons with regular arrangements,deep stains,and normal forms.These results showed that DZSM could significantly improve the neuronal function of APP/PS1 mice.The state of oxidative stress was detected by oxidative stress kit,MDA activities of APP/PS1 mice were up-regulated,while SOD and GSH-Px activities were down-regulated.After DZSM treatment,MDA activities were significantly decreased,while SOD and GSHPx activities were significantly increased.These results showed that DZSM alleviated oxidative stress in APP/PS1 mice.The state of cholinergic system was detected by acetylcholine kit,the AChE activities of APP/PS1 mice were up-regulated,and the activities of ACh and ChAT were downregulated.After DZSM treatment,the AChE activities were significantly decreased,and the activities of ACh and ChAT were significantly increased.These results showed that DZSM could relieve the damage of cholinergic system in APP/PS1 mice.TUNEL staining results showed that the number of TUNEL-positive cells in the cortex and hippocampal(CAI)of APP/PS1 mice was significantly increased,and the fluorescence intensity was significantly up-regulated.After DZSM treatment,the number of TUNELpositive cells was significantly decreased,and the fluorescence intensity was significantly decreased.Western blot results showed that the expression of Bax in APP/PS1 mice was up-regulated,the expression of Bcl-2 was down-regulated,and the ratio of Bax/Bcl-2 was significantly increased.After DZSM treatment,the expression of Bax was down-regulated,the expression of Bcl-2 was up-regulated,and the ratio of Bax/Bcl-2 was significantly decreased.These results showed that DZSM inhibited apoptosis in APP/PS 1 mice.3.Effect of DZSM on endoplasmic reticulum stress of APP/PS1 transgenic miceWestern blot and immunofluorescence results showed that the levels of PERK pathway proteins in APP/PS 1 mice were up-regulated,including P-PERK/PERK,P-eIF2α/eIF2α,ATF4,CHOP and QRICH1.After DZSM treatment,the levels of PERK pathway proteins were significantly decreased.The levels of IRE1α and ATF6 pathways in APP/PS1 mice were up-regulated,including P-IRE1α/IRE1α,XBP1s and ATF6,and the levels of IRE1αand ATF6 pathways were significantly decreased after DZSM treatment.These results suggested that DZSM might play a neuroprotective role against AD by inhibiting ER stress pathways.32 active components of DZSM were screened out from the TCMSP and ETCM online database,PubChem and SwissTargetPrediction platform,and then molecular docked with QRICH1,respectively.The binding energies were all less than-1.2kcal/mol,indicating that QRICH1 might be a potential target for DZSM to exert anti-AD effects.Among the 32 active ingredients,poriferasterol and beta-sitosterol were most firmly combined with QRICH1.indicating that poriferasterol and beta-sitosterol might be the effective components of DZSM targeting QRICH1.4.Effect of DZSM on Aβ1-42-induced HT22 cell injury and its mechanismThe results of CCK8 showed that DZSM medicated serum significantly increased the cell viability of HT22 cells induced by 10μM Aβ1-42,and played a neuroprotective role.Western blot results showed that in Aβ1-42 group,the protein levels of PERK pathway(including P-PERK/PERK,P-eIF2α/eIF2α,ATF4,CHOP,and QRICH1),IRE1α pathway(including P-IRE1α/IRE1α,XBP1s)and ATF6 pathway were up-regulated.The protein levels of the three pathways were significantly decreased after treatment with DZSM medicated serum.These results suggested that DZSM could inhibit ER stress pathway in Aβ1-42 induced HT22 cells.ConclusionThis study preliminarily elucidated the neuroprotective effects and potential mechanism of DZSM in the treatment of AD.In vitro and in vivo experiments revealed that DZSM could inhibit ER stress pathway,reduce Aβ plaque accumulation and neuronal injury,and improve the learning and memory impairment of APP/PS1 transgenic mice.Besides,network pharmacology and molecular docking have suggested that QRICH1 might be one of core target for DZSM to exert anti-AD effects.