Protective Effect And Mechanism Of Ginsenoside Rg1 Against Cognitive Impairment By Inhibiting TRPC6 Channel In T2DM Mice

ObjectiveTo evaluate the protective effect of Rg1 against cognitive dysfunction in T2DM mice,and its effects on neuronal damage,neuroinflammation,and calcium homeostasis;Further explore the effect of TRPC6 knockout against cognitive dysfunction in T2DM mice and the underlying mechanism by which Rg1 functions.Methods1.C57 BL/6 mice were divided into normal control group（Control）,high-fat diet group（HFD）,model group（HFD+STZ）,ginsenoside Rg1（1 mg/kg,5 mg/kg and 10 mg）/kg),positive drug metformin 200mg/kg group（Metformin）.After 8 weeks of high-fat feeding,mice were given 110 mg/kg STZ solution by intraperitoneal injection.Successfully established model mice were treated with corresponding drugs for 8weeks,and the changes in body weight,blood glucose,food intake,and water intake were recorded continuously.The changes in spontaneous activity and exploratory behavior of T2DM mice were observed by open field test.The changes in learning and spatial memory ability of T2DM mice were detected by morris water maze test.Histological staining such as HE staining,Nissl staining,and β-gal staining was used to detect neuronal lesions and aging in the cerebral cortex and hippocampus of T2DM mice;Prussian blue and DHE staining were used to observe the accumμlation of ROS and the vascμlar lesions in the brain of T2DM mice;Aβ1-42 immunofluorescence staining were used to investigate the generation of Aβoligomers in the brain of T2DM mice.Western Blot and RT-PCR were used to detect the changes of PSD95,APP,BACE,CTF-β,NCSTN,Aβ1-42,p-Tau,NOX2,p22phox,p47phox,CD36,TRPC6,CN,NFAT1,NLRP1,ASC,caspase-1,IL-1βin protein and m RNA in the brain tissue.2.TRPC6^-/-mice and littermate WT mice were divided into 5 groups:WT group,WT+HFD+STZ group,TRPC6^-/-group,TRPC6^-/-+HFD group,and TRPC6^-/-+HFD+STZ group.The mice in the WT and TRPC6^-/-groups were fed their growth maintenance diet,while the mice in the other groups were fed a high-fat diet and had unlimited access to food and water.After 8 weeks of high-fat eating,mice were given an intraperitoneal injection of 110 mg/kg STZ solution.To determine whether the modeling was successful,fasting blood glucose was checked 72 hours later.TRPC6knockout T2DM mice had their physiological changes evaluated by recording and assessing changes in body weight,blood glucose,food and water intake.OFT was utilized to investigate changes in T2DM mice’s spontaneous activity and exploratory behavior.Learning and spatial memory abilities in TRPC6 knockout T2DM mice were assessed by MWM and CFC tests.OGTT test was used to measure mouse glucose tolerance before sampling.Neuronal lesions and aging changes in the cerebral cortex and hippocampus of T2DM mice were studied using histological staining such as HE staining,Nissl staining,β-gal staining,and so on.Subsequently,DHE staining was used to analyze ROS accumulation in the brain of T2DM mice.Aβ1-42immunofluorescence staining were used to explore the generation of Aβoligomers in the brain of T2DM mice;Western Blot and RT-PCR were used to detect proteins and m RNA expression changes of APP,BACE,NCSTN,NOX2,p22phox,p47phox,CD36,TRPC6,CN,NFAT1,NLRP1,ASC,caspase-1,IL-1β.Resμlts1.Rg1 has a blood sugar-lowering effect,but it is not as effective as Metformin.The moving distance,average moving speed,lines crossing,and exploratory behaviour of T2DM mice have deteriorated to some extent as the disease progresses,and Rg1 and metformin treatment can improve behavioural performance in the open field test.The MWM experiment revealed that Rg1 treatment increased T2DM mice’s learning and memory ability,and that Rg1’s effect was superior to that of metformin.Histological staining by HE and Nissl showed that Rg1 improved neuronal damage,ageing,and cerebrovascular lesions in T2DM mice.The results of PSD95 immunofluorescence and WB showed that Rg1 and metformin treatment improved synaptic damage in the brain of T2DM mice.Aβ1-42 immunofluorescence staining showed that Rg1 treatment decreased the number of amyloid deposits in the brain.WB and RT-PCR results showed that Rg1 treatment reduced the protein expression of NCSTN,Aβ1-42,p-Tau,NOX2,p22phox,p47phox,CD36,TRPC6,CN,NFAT1,NLRP1,ASC,caspase-1,IL-1βand related m RNA expression.Rg1 reduced the concentrations of IP3 and DAG in the brain of T2DM mice,according to Elisa experiment using brain tissue homogenate.Moreover,Rg1 reduced the concentration of intracellular Ca²⁺in the brain tissue.2.TRPC6 knockout has a protective effect on weight loss and blood sugar increase in T2DM mice.OFT and MWM test showed that TRPC6 knockout coμld improve the exploratory behavior,learning and memory ability of T2DM mice.Histological staining HE and Nissl showed that TRPC6 knockout improved neuronal damage and aging in T2DM mice.TRPC6 knockout ameliorated synaptic damage in the brain of T2DM mice according to PSD95 immunofluorescence and Western blot.Aβ1-42immunofluorescence staining both showed that TRPC6 knockout treatment improved amyloid deposition in the brain.WB and RT-PCR results showed that TRPC6 knockout reduced the protein expression of NCSTN,Aβ1-42,p-Tau,TRPC6,CN,NFAT1,NLRP1,ASC,caspase-1,IL-1βand related m RNA in the brain of T2DM mice,but have no effect on NOX2,p22phox,p47phox,CD36 expression.At the same time,TRPC6 knockout reduced the concentration of Ca²⁺in brain tissue of T2DM mice.ConclusionRg1 can improve cognitive dysfunction in T2DM mice,and knockout of TRPC6 gene also reduced neuroinflammation,Aβdeposition and calcium overload,improving T2DM-induced cognitive dysfunction.Rg1 playing a cognitive protective role by inhibit NOX2-TRPC6-NLRP1 signaling pathway,and TRPC6-mediated Ca²⁺is a potential therapeutic target for cognitive dysfunction in T2DM.