An Experimental Study On The Connotation Of "Intrinsic Deficiency" In Cognitive Impairment Caused By Aging Based On Carnosine Metabolism

ObjectiveThere are many reasons for cognitive impairment caused by aging,and it is believed that the focus is on "deficiency in origin".The causes include deficiency of kidney essence,deficiency of qi and blood,and deficiency of the five internal organs.Studies have found that the content of endogenous anti-oxidant small molecule carnosine in the elderly is much lower than that of adults,and it also belongs to the category of "deficiency in origin".According to preliminary laboratory research and related literature reports,carnosine can play a neuroprotective effect.In this project,HT22 cells induced by the AAPH and senescence-accelerated mouse prone 8(SAMP8)are selected as experimental models in vitro and in vivo.In exploring whether carnosine can play the same beneficial effects in cognitive impairment caused by aging,and the mechanism behind it.Methods(1)In vitro experiment:HT22 cells were selected as experimental objects,and AAPH was used to induce oxidative damage of cells.The experiment was divided into control group,normal administration group,model group and administration group.Observe the cell morphology under a microscope to determine its survival state.The thiazolyl blue[2,2’-azobis(2methylpropionamidine)dihydrochloride,MTT]method detects cell viability;the kit detects the level of reactive oxygen species(ROS);antioxidant Enzyme superoxide dismutase(SOD)activity,lipid oxidation reaction end product malondialdehyde(Methane dicarnosineboxylic aldehyde,MDA)level;Tunel and Hoechst 33342 staining to detect cell apoptosis;western bloting to detect SIRT6 and NLRP3 inflammasome pathway related proteins(NLRP3,ASC,Caspase-1,IL-18,IL-1β),apoptosis-related proteins(Bax,Bcl-2,Caspase-3);qRT-PCR detection of sirtuins family expression;through the Maestro 11.8 software and the CB-Dock website discuss the docking of carnosine and SIRT6,and evaluate their combined ability.(2)In vivo experiment:9-month-old SAMP8 mice were selected as experimental animal models.The experiment was divided into control group,model group,low-dose administration group(carnosine 100 mg/kg),and high-dose administration group(carnosine 200 mg/kg).The mice were intragastrically administered with normal saline and different doses of carnosine once daily for six weeks.Evaluate the cognitive ability of mice through the behavioral open field experiment and new object recognition experiment;the kits detect the ROS level,SOD activity,MDA level and adenosine 5’-triphosphate(ATP)content in the hippocampus and cortex tissues of the mice;western bloting detects the expression of SIRT6,mitochondrial-related proteins(Mitofusin-1,Mitofusin-2,Drp1,P-Drp1),the expression of inflammasome pathway related proteins(NLRP3,ASC,Caspase-1,IL18,IL-1β),and the expression of apoptosis-related proteins(Bax,Bcl-2,Caspase-3).Results(1)In vitro experiment:After the free radical inducer AAPH treats the cells,the survival rate of neuronal cells is significantly reduced.100mM carnosine can improve the damage of APPH to neuronal cells and increase the survival rate of cells;reduce the level of reactive oxygen species and the content of lipid peroxidation products,and increase superoxidation The activity of biological dismutase can improve the body’s antioxidant capacity,reduce cell apoptosis,and promote nerve cell survival;promote the expression of longevity protein SIRT6,and improve the activation of NLRP3 inflammasome;molecular docking results suggest that the small molecule carnosine binds to the active pocket of protein SIRT6,The combination of the two is relatively close.(2)In vivo experiment:Behavioral results show that carnosine has the ability to improve cognitive impairment.In the open field experiment,carnosine increased the distance to the central area of SAMP8 mice.In the new object recognition experiment,the recognition rate of new objects is improved;the expression of mitochondrial fusion protein(Mitofusin-1,Mitofusin-2)is increased,and the expression of mitochondrial division protein(Drp1,P-Drp1)is decreased,maintaining the integrity of mitochondrial structure and increasing ATP Content:Reduce ROS level and MDA level,improve SOD activity,improve body’s antioxidant capacity,reduce cell apoptosis and inflammation,and promote nerve cell survival.ConclusionsIn the cognitive impairment caused by aging,the exogenous supplementation of carnosine has a significant neuroprotective effect.It can reduce the oxidative damage caused by AAPH in vitro,increase the activity of antioxidant enzymes,and reduce inflammation and apoptosis of neuronal cells;in vivo,it can significantly improve cognitive decline caused by aging.This neuroprotective effect is related to the improvement of mitochondrial function and the activation of SIRT6.