| [Objective]To establish a rat model of type 2 diabetes mellitus(T2DM)by high-sugar and high-fat diet and Streptozotocin(STZ,35mg/kg)to investigate whether different doses of Scutellarin(Scu)can regulate BDNF/TrkB and PI3K/Akt/CREB signaling pathways by inhibiting inflammatory factors and oxidative stress,alleviating hippocampal neuronal apoptosis in the hippocampus and ameliorating diabete-associated cognitive decline via the intragastric therapy.[Methods]1.Ten-week-old male Sprague-Dawly rats(SD;n=100,weighing 160±200g)were fed adaptively for two weeks,and then 15 rats were divided into control group and the rest were selected as T2DM group according to completely randomized digital table.Rats in the Control group were fed with standard diet and rats in the T2DM group were fed with high-glucose and high-fat diet.8W later,rats in the T2DM group were given fasting intraperitoneal injection of 35mg/Kg STZ all at once to induce T2DM.Three days later,the random blood glucose concentration in the tail vein of SD rats was determined.If the blood glucose concentration was≥ 16.7mmol/L and maintained for more than 1W,the model of type 2 diabetes mellitus was established successfully.2.According to completely randomized digital table,the rats in the T2DM group were divided into the model group(Model),the Scu groups(Scu25,50,100 mg/Kg,respectively),and the metformin group(Met,120mg/Kg).Meanwhile,the rats in the control group and the model group were given the same volume of 0.9%normal saline(NS).Drugs were given via oral gavage once daily.It lasts eight weeks.During the period of intragastric administration,the changes of general condition,body weight and fasting blood glucose(FBG)in each group were monitored weekly,and the volume of intragastric administration was adjusted according to body weight.At the end of the 4W and the 8W of intragastrial administration,oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)were performed to detect blood glucose homeostasis,respectively.3.Morris water maze(MWM)and Open field test were performed to identify the spatial learning and memory performances,explore behavior and tension in a novel environment after intragastric administration among the control and diabetic rats.4.After the behavioral experiment,Fasting blood glucose(FBG)and Fasting insulin(FINS)were measured by Enzymelinked immunosorbent assay(ELISA),and the insulin resistance index(HOMA-IR)and insulin sensitivity index(ISI)of the rats were calculated accurately.5.Blood lipid and hepatic biochemical indexs in serum were measured,including alanine aminotransferase(ALT),aspartic transaminase(AST),total cholesterol(TC),and triglyceride(TG).6.ELISA was used to measured Malondialdehyde(MDA),Reactive oxygen species(ROS),Catalase(CAT),Glutathione(GSH),Superoxide dismutase(SOD)in serum and hippocampus tissue.7.Tumor necrosis factor-α(TNF-α),Interleukin-1β(IL-1β),Interleukin-6(IL-6)in serum and hippocampus tissue were detected by ELISA.8.Hematorylin and eosin staining(HE),Nissl staining and TdT-mediated dUTP nick end labeling staining(Tunel)were performed to observe the pathological changes of neurons in the hippocampus tissue of rats.9.Immunohistochemistry(IHC)and Western blotting(WB)were used to detect brain-derived neurotrophic factor/trk receptor tyrosine kinase B(BDNF/TrkB)in hippocampus of rats and its downstream signaling pathway phosphatidylinositol 3 kinase/protein kinase B/cAMP response element binding protein(PI3K/Akt/CREB),caspase family members(procaspase-3,Cleaved caspase-3,procaspase-8,Cleaved caspase-8,procaspase-9,procaspase-9,procaspase-12)and B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)also be examined.[Results]1.The model of T2DM in SD rats was successfully established by high-glucose and high-fat diet+low dose of STZ intraperitoneal injection.During gavage treatment,the body weight of rats in the control group increased steadily,and blood glucose was stable in the normal range.Compared to the control group,the body weight of rats in model group continued to decrease,blood glucose significantly increased,glucose tolerance and insulin tolerance were seriously impaired(P<0.05).Each Scu treatment group rats weight was still on the decline,but the degree of decline leveling off,there is time dependent and dose dependent.Blood sugar,glucose tolerance and insulin tolerance in 4W and 8W,insulin tolerance in 8W not seen obviously improved(P>0.05).2.Morris water maze(MWM):(1)Compared with the control group,the model group showed severe spatial learning impairment,which was characterized by prolonged escape latency(P<0.05);Compared with Model group,the escape latency of Scu groups and the Met-120 group was significantly shortened(P<0.05).With the increase of training times,the total distance of each group decreased gradually.From day 2 to day 5,compared with the control group,the total distance of rats in model group was prolonged(P<0.05);Compared with Model group,the total distance of Scu groups and the Met-120 group was shortened on day 2-4(P<0.05).On day 5,the total distance was shortened,but the difference was statistically significant only in Scu-50 group(P<0.05).(2)The results of space exploration showed that there was no significant difference in total distance and average velocity of rats among all groups(P>0.05).Compared with the control group,the number of crossing platforms,the percentage of target quadrant distance and the percentage of target quadrant residence time of rats in the model group were decreased(P<0.05);Compared with the model group,the number of crossing platform,the percentage of target quadrant distance and the percentage of target quadrant residence time in Scu-25,Scu-50,Scu-100 groups and the Met-120 groups were increased(P<0.05),and the effect of Scu in the Scu-50 group was better than that of other groups.Open field test(OFT):Compared with the control group,the total exploration distance,the average velocity,number of times enter the central area,times spent in the central area,number of vertical movements were shortened and the number of grooming increased in the model group,and the differences were statistically significant(P<0.05).Compared with the model group,the total exploration distance,the average velocity,number of times enter the central area,times spent in the central area,number of vertical movements were longer,and the number of grooming increased in the Scu-25,Scu-50,Scu-100 and Met-120 group.The differences were statistically significant(P<0.05).Among them,the effect of Scu in the Scu-50 group was better than that of other groups.3.Compared with the control group,the levels of FBG,FINS and HOMA-IR in the model group were increased and the level of ISI in the model group were decreased(P<0.05);Compared with the model group,the levels of FBG,FINS,HOMA-IR in Scu groups and the Met group were decreased and the level of ISI in the Scu group were increased(P<0.05).4.Blood lipid and hepatic biochemical indexs in serum were shown that compared with the control group,the levels of ALT,AST,TC and TG in serum of rats in the model group were increased(P<0.05);However,compared with the model group,the levels of ALT,AST,TC and TG in Scu groups and the Met group were decreased(P<0.05).Among them,the effect of Scu in the Scu-100 group was better than that of other groups.5.Compared with the control group,in the serum,hippocampus tissue of the model group rats,oxidative stress related factors(MDA and ROS)levels were increased(P<0.05),anti oxidative stress related factors levels(CAT,GSH and SOD)were decreased(P<0.05),inflammatory factors(TNF-α,IL-1β,IL-6)were increased(P<0.05);Compared with the model group,after Scu and Met treatment,in the serum,hippocampus tissue of the model group rats,oxidative stress related factors(MDA and ROS)were decreased(P<0.05),anti oxidative stress related factors(CAT,GSH and SOD)levels increased(P<0.05),inflammatory factors(TNF-α,IL-1β,IL-6)were decreased(P<0.05),there is dose dependent and in the Scu-25 group there was no statistically significant difference in some of the indicators(P>0.05).6.HE staining,Nissl staining and TUNEL staining revealed that in the hippocampus tissue of Model group rats,the nucleus of neure was karyopyknosis and densely stained,the number of Nissl bodies was decreased.Moreover,there are numerous yellowish-brown apoptotic bodies.These results indicated that there were obvious pathological damage of neurons in the model group.After Scu and Met treatment,the phenomenon of karyopyknosis and dense staining was reduced,the number of Nissl bodies and apoptotic bodies was increased.These results indicated that Scu can improve DACD rat hippocampus tissue neuron injury.7.The results of IHC and WB showed that compared with the control group,the protein expressions of BDNF,p-TrkB,TrkB,p-PI3K,PI3K,p-Akt(Ser473),p-Akt(Thr308),Akt,p-CREB,CREB and Bcl-2 in the hippocampus tissue of rats in the model group were down-regulated,the differences were significant(P<0.05).After drug treatment,the protein expression levels of BDNF,p-TrkB,TrkB,p-PI3K,PI3K,p-Akt(Ser473),p-Akt(Thr308),Akt,p-CREB,CREB and Bcl-2 were up-regulated,and the differences were significant(P<0.05).8.IHC and WB results showed that,compared with the control group,in the model group,the protein expression of Procaspase-3,Cleaved-caspase-3,Procaspase-8,Cleaved-caspase-8,Procaspase-9,Cleaved-caspase-9,Procaspase-12,and Bax in the hippocampus tissue of rats were increased,and the differences were significant(P<0.05).After Scu treatment,protein expression levels of Procaspase-3,Cleaved-caspase-3,Procaspase-8,Cleaved-caspase-8,Procaspase-9,Cleaved-caspase-9,Procaspase-12 and Bax in the hippocampus tissue of rats were decreased,and the differences were statistically significant(P<0.05).[Conclusions]1.Scu can reduce the expression imbalance of inflammatory factors,anti-oxidative stress,regulate lipid metabolism disorders,and ameliorate insulin resistance.2.Scu can reduce the apoptosis of neurons in hippocampus tissue of T2DM rats by up-regulating BDNF/TrkB and its downstream signaling pathway PI3K/Akt/CREB,thus improving the learning and memory ability of rats.Scu play a neuroprotective role by alleviating the pathological damage. |
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