| Alzheimer’s disease(AD)is a kind of neurodegenerative disease mainly characterized by learning and memory disorder,which is the severe medical and social problem encountering in the 21st century.Its features are cognitive impairment,behavioral abnormality and personality change.And the symptoms are frequently aggravated with age,which develop from the mild memory impairment gradually to severe dementia,and its average course of disease with incubation period is about 7-10 years.AD is extremely common for the aging population in the modern society,lacking of effective therapeutic strategy at present,which has become one of the important causes of elderly death.Notably,AD has severely damaged the elderly health and imposed tremendous burdens on the family and the society.Therefore,how to control and treat AD has become a pressing global research topic.Oxidative stress is an important factor that plays a potential role in the occurrence of neurodegenerative disease,which has long been paid a great deal of attention from scientists.In recent years,researchers have gradually focused on the neuroprotection and antioxidation activities of traditional Chinese medicine,and attempted to search for the entry for preventing and treating AD.Comi Fructus is the dry mature fruit of Cornus oficinalis Sieb.et Zucc..The medicinal herb,which was first reported in Shen Nong’s Herbal Classic to possess liver and kidney tonifying,as well as seminal emission arresting and desertion relieving.It is the famous traditional Chinese medicine in China,as well as one of the major components in famous traditional Chinese medicine like Liuwei Dihuang Pills,Kidney-qi-tonifying pills and Zuogui pills.Cornuside is one of the major cornel iridoid glycosides in cornus officinalis.The pharmacological research on Cornuside is rare,and only several studies have respectively reported its anti-inflammation,anti-apoptosis and improvement of mitochondrial dysfunction.According to our previous study,Comuside could suppress the H2O2-induced oxidative damage in SH-SY5Y cells.However,there are no relevant reports on the cognitive impairment induced by oxidative stress,let alone relevant studies to investigate its mechanism and target,while Corni Fructus and its cornel iridoid glycosides have been reported to exert beneficial effects on antioxidant activity and the improvement of cognitive impairment.Therefore,we decided to carry out research on the effect of Cornuside on improving cognitive disorder and the related molecular mechanism from the point of the oxidative stress.Part 1 Protective effect of Cornuside on SH-SY5Y cell injury induced by H2O2 Objective To explore the effect of the active compound Cornuside which is isolated from the Chinese medicine Cornus officinalis Sieb.et Zucc.on the growth of SH-SY5Y cells and its neuroprotective effect on SH-SY5Y cell injury induced by H2O2.Method The SH-SY5Y cells were pre-treated with Cornuside(0.3,1,3 μmol/L)for 2 h,then they were added with H2O2 300 μmol/L for 12 h to induce oxidative damage of the SH-SY5Y cells.After that,cell morphology was observed under microscopic conditions;CCK8 method was used to detect cell survival rate.DPPH method was applied to measure anti-oxidative activity of Cornuside in vitro.LDH release rate was measured by spectrophotometer method;mitochondrial membrane potential was detected by JC-1 assay.Fluorescence microscope was chosen to detect intracellular ROS level.Furthermore,cell apoptosis was detected by Hochest 33258 and AO/EB staining.Moreover,the activity of intracellular SOD,GSH level and MDA content were measured by spectrophotometer method.Results Our research discovered that Cornuside had distinct scavenging effect on free radicals,with the EC50 value of 2.01 ×10-5 mol/L,and Cornuside had no toxic effect on normal SH-SY5Y cells under the concentration of 0.01-30μmol/L.Compared with the model group,Cornuside not only increased cell survival rate in a concentration-dependent manner at 0.3,1,3 μmol/L but also changed the damaged cell morphology and apoptosis level obviously.In addition,Cornuside could reduce LDH leakage rate and increase mitochondrial membrane potential level.It also increased the levels of SOD and the GSH as well as reduced the production of MDA.Overall,Cornuside exerted the greatest medicinal effect at the concentration of 1 μmol/L,and the anti-oxidative effect is equivalent to VE.Part 2 Pharmacological research of Cornuside on mice with learning and memory impairment induced by D-galactose in vivoObjective To explore the effect of the active compound Cornuside which is isolated from the Chinese medicine Cornus officinalis Sieb.et Zucc.on the cognitive impairment caused by oxidative stress.Method D-galactose model was adopted,which was closely related to oxidative-stress damage and affected the function of learning and memory.According to the body weight,two-month-old male KM mice were randomly divided into Control,Model,Cornuside(3,12.5,50 mg/kg)groups,positive drug donepezil(1 mg/kg)and Huperzine A(0.1 mg/kg)groups,with 15 mice in each group.Mice in model and administration groups were injected with 100 mg/kg D-Gal subcutaneously on the nape of the neck,and for those in control group,an equal volume of saline was injected subcutaneously at the same location.Mice in all groups were given the continuous gavage for eight weeks.And from the 9th week,behavioral tests(such as Autonomous activity test、Morris water maze test and step-through test)were conducted to observe the behavior of mice.The degree of oxidative damage was determined by the levels of SOD,MDA,GSH-Px,T-AOC in serum and AGEs in the brain.The degree of damage to the cholinergic system was measured by the Ach content and AChE level in the brain.We also used the the secretion levels of inflammatory factors such as NO,IL-6,IL-1β and TNF-α in the brain to evaluate the degree of inflammatory response.Results Our research found that compared with mice in the Control group,the model group’s ability of moving autonomously was not affected.Compared with mice in the model group,according to Morris water maze and step-through tests,we discovered that Cornuside remarkably improved the ability of spatial memory and memory reappearance of the model mice which was manifested as shortened time to search for platform,increased frequency and enhanced capacity to avoid electrical shock of mice after administrating Cornuside.We also discovered that Cornuside possessed anti-oxidative damage effect,which was manifested as the increased levels of SOD,T-AOC,GSH-Px and decreased content of MDA in serum,along with the remarkably decreased AGEs contents in hippocampus and cerebral cortex.Furthermore,Cornuside acted on the cholinergic system and inhibited the expression of AChE in the brain.Finally,we discovered that Cornuside suppressed the secretion of inflammatory factors(such as NO,IL-6,IL-1β and TNF-α)in hippocampus and cerebral cortex.Above indicators are significantly different from the model group(P<0.05 or P<0.01).Typically,the optimal Cornuside effect was achieved at the medium dose of 12.5 mg/kg.Part 3 The mechanism research of Cornuside on mice with learning and memory impairment induced by D-galactose in vivoObjective To explore the preliminary mechanism of the active compound Cornuside which is isolated from the Chinese medicine Cornus officinalis Sieb.et Zucc.on mice with learning and memory impairment induced by D-Gal.Method According to the body weight,two-month-old male KM mice were randomly divided into Control group,Model group,Cornuside(3,12.5,50 mg/kg)groups,positive drug donepezil(1 mg/kg)and Huperzine A(0.1 mg/kg)groups,with 15 mice in each group.Mice in model and administration groups were injected with 100 mg/kg D-Gal subcutaneously on the nape of the neck,and for those in control group,an equal volume of saline was injected subcutaneously at the same location.Mice in all groups were given the continuous gavage for eight weeks.After that the effect of Cornuside on the loss of neurons in the hippocampal CA1 and CA3 regions of mice induced by D-Gal were detected by HE and Nissl staining.Immunohistochemistry method was used to detect the expression levels of RAGE and BDNF in cerebral cortex as well as GFAP and Iba 1 in hippocampus.Western blot was used to investigate the expression and phosphorylation levels of key proteins,including IκBa,JNK,ERK1/2 and p38 in the classic inflammation AP-1 and NF-κB signaling pathways.Finally,the target protein was screened by Western blot,and molecular docking was used to verify the target.Results Firstly,HE staining and Nissl staining suggested that Cornuside remarkably increased the number of neurons in hippocampal CA1 and CA3 regions in mice as well as the number of Nissl body.Secondly,immunohistochemistry staining results showed that Cornuside up-regulated the expression levels of BDNF and down-regulated the RAGE and Iba 1 in cerebral cortex as well as GFAP in hippocampal CA1 and CA3 regions.Meanwhile,it is reported in numerous studies that neuronal injury is regulated by neuroinflammation.Therefore,Western blot assay was conducted to check the AP-1 and NF-κB classical inflammatory signaling pathways.Subsequently we discovered that Cornuside inhibited the phosphorylation of ERK1/2 in the AP-1 signaling pathway and IκBa in the NF-κB signaling pathway,thus reducing the secretion of related inflammatory factors.But it had no obvious inhibitory effect on the phosphorylation of JNK and p38 in the AP-1 signaling pathway.Finally,molecular docking technology was used to verify the target of IκBα and ERK1/2.Results showed that Cornuside bound weakly to IκBα but strongly to ERK1/2.Based on the above researches,this paper could draw the following preliminary conclusions:1.Cornuside could play a neuroprotective role in H2O2-induced SH-SY5Y cell injury which might be related to the mechanism of decreasing oxidative damage and inhibiting neuronal apoptosis at a concentration that does not affect the viability of SH-SY5Y cells.2.Cornuside significantly improves the learning and memory function on mice with learning and memory impairment induced by D-Gal.This effect may be achieved by reducing the oxidative stress state and the expression level of inflammatory factors in brain injured mice.3.The neuroprotective effect of Cornuside may be achieved by regulating the AGEs-RAGE-IκBα-ERK1/2 signaling pathway.Cornuside can also improve the abnormal phosphorylation state of the related proteins,and and the key protein could be the ERK1/2.To sum up,this study applies the H2O2-induced SH-SY5Y neuronal cell model and the D-Gal-induced learning and memory impairment model,to explore the neuroprotective effect of Cornuside in vivo and in vitro respectively.And the differences between different doses were analyzed and clarified,laying a foundation for further researches.Notably,this study is the first to verify that Cornuside,the cornus officinalis iridoid glycoside compound,possesses favorable anti-oxidative damage,anti-neuroinflammation,and anti-neuronal apoptosis effects,thus exerting multiple protective effects on improving cognitive disorder.Such effect may be achieved through regulating the AGEs-RAGE-IκBα-ERK1/2 signaling pathway.The above experimental results have provided direct experimental foundation for the prevention and treatment of AD through Cornuside.In addition,our results also reveal that medicinal materials or foods rich in Cornuside can be potentially used to treat oxidative stress and inflammation-related diseases.Moreover,this study determines that ERK may be one of the Cornuside targets,which also reminds us that Cornuside has potential research value in other diseases involving the ERK signaling pathway. |
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