Effect And Mechanism Of Forsythoside B On Cognitive Function In Menopausal Diabetes Mellitus

Part one Analysis of relevant risk factors for cognitive dysfunction in diabetesObjective: To study the changes of cognitive function and the related factors in patients with type 2 diabetes.Methods: Based on the criteria,76 patients with type 2 diabetes who enrolled to the endocrinology department of Tangshan Workers’ Hospital from September 2020 to September 2021 were collected in this study.We divided them into the cognitive normal group of type 2 diabetes and the cognitive dysfunction group of type 2 diabetes according to the results of mini-mental state examination.Subsequently,comparison of general data,laboratory test indicators and the scores of repeatable battery for the assessment of neuropsychological status between the two groups were evaluated.And Logistic binary regression analysis was applied to explore the risk factors of cognitive dysfunction in patients with type 2 diabetes.Results:There were significant differences in age,gender,diseases duration,education,and combining coronary heart disease,hypertension,diabetic retinopathy and diabetic nephropathy between the two groups(P<0.05);Fasting blood glucose levels were significantly higher in cognitively impaired type 2 diabetic patients than in cognitively normal type 2 diabetic patients(P<0.05);Vision,language ability,attention,immediate memory,delayed memory,total score with type 2 diabetic cognitive dysfunction were significantly reduced compared with type 2 diabetes patients with normal cognitive function(P<0.05);Total scores on a repeatable test assessing neurobehavioral status in patients with type 2 diabetes gradually decreased with age(P<0.05);The total score of repeatable battery for the assessment of neuropsychological status in the female patients with type 2 diabetes was significantly reduced compared with male patients(P<0.05);Comprehensive reproducible test results to assess patient neuropsychological status with disease duration of type 2 diabetes greater than 20 years were significantly lower compared with patients with disease duration of type 2 diabetes less than 10 years(P<0.05);Age,gender and combing with diabetic retinopathy were found to be risk factors for cognitive impairment in type 2 diabetes(P<0.05).Part two Experimental study on effects of oophorectomy on cognitive function in diabetic miceObjective: In this study,ovariectomy(OVX)is to be performed in diabetic mice to investigate the effects of ovariectomy on cognitive dysfunction in diabetic mice.Methods: Sixty 8-week-old female C57BL/6J mice were selected and randomly divided into 4 groups(n=15/group):control group(sham operation),diabetes model group(DM),ovariectomy group(OVX),ovariectomy group(O+D)).OVX was performed on 8-week-old mice,and mice in DM group and O+D group were injected with streptozotocin(60mg/kg/day/L)and citrate buffer for 5 consecutive days one week after surgery to induce DM model.The success of membrane building was indicated by blood glucose of mice greater than 16.6mmol/L.Mouse tail venous blood was taken for glucose tolerance test.After 8 weeks,the animals in each group were subjected to water maze and Y maze experiments.After the behavioral experiment,the animals were euthanized and sampled,and the hippocampus of the mice was kept.Six hippocampi were collected from each group for decalcification,embedding,sectioning and staining,and six hippocampi were collected for freezing preservation.HE staining and Nith staining were used to observe the morphological changes of hippocampus.Results:1 Behavioral experiment results1.1 Water Maze results: Compared with Sham group,OVX,DM and O+D groups showed more chaotic motion,longer escape delay and reduced number of platform crossing.And,compared with Sham group,the target quadrant time in OVX,DM and O+D groups was significantly shortened.Among them,O+D group had the most significant differences.In addition,there was no significant difference in swimming speed between these groups.1.2Y Maze results: Compared with Sham group,OVX,DM and O+D groups had less spontaneous alternating motion(FIG.3A).Among them,the O+D group had the least spontaneous alternating motion.2 Histological resultsThe results of hematoxylin-eosin staining and Nissl staining showed that the hippocampal morphology of the Sham group was normal,with obvious nucleoli and clear nuclear membranes.While OVX,DM and O+D groups had loss of hippocampal neurons,sparse arrangement of hippocampal cells and blurred boundaries.The O+D group was the most severely affected.Part three Study on the effect and mechanism of forsythoside B on neurocognitive degeneration in ovariectomized diabetic miceObjective: In this study,forsythoside B(FTS·B)can delay Ovariectomy(OVX)in mice with diabetes mellitus(DM),however,its mechanism of action has not yet been investigated.We intend to apply the model of serious functional degeneration induced by ovarian resection combined with diabetes mellitus.To further investigate the mechanism of FTS·B and estrogen 17β-estradiol(ER)on neurocognitive degeneration in diabetic mice induced by ovariectomy.Methods: Seven days after bilateral ovariectomy(OVX)or sham surgery,adult female C57BL/6J mice(n=15/group)received intraperitoneal injection of streptozotocin(60mg/kg/day/L)and citrate buffer for 5 consecutive days to induce diabetes mellitus(DM).Fourteen days later,ovariectomized diabetic mice were given intraperitoneal injection of FTS·B(100,150 mg/kg/day/L)and subcutaneous injection of 17β-estradiol(1mg/kg)for 8weeks[OVX+DM+low-FTS·B group(L-F),OVX+DM+high-FTS·B group(H-F),and OVX+DM+17β-estradiol(ER)].In addition,the following control groups were defined: Sham,OVX,DM,and OVX+DM(O+D).Fasting plasma glucose,body weight and blood insulin levels were determined in each group of mice.Next,their cognitive function was tested through behavioral experiments.Morphological changes in the hippocampus were demonstrated by hematoxylin and eosin(H&E)staining and Nissl staining.Immunohistochemistry confirmed β-amyloid(Aβ)aggregation and p-tau hyperaggregation.Interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),brain-derived neurotrophic factor(BDNF),postsynaptic density-95(PSD-95),synaptophysin,and synapsin-1 expression in the hippocampus was detected by real-time polymerase chain reaction(RT-PCR)and western blot analysis.Results:1 Effects of FTS·B on blood glucose,body weight and blood insulin level(FBI)in ovariectomized diabetic mice.Fasting glucose levels and blood insulin levels were measured in each group,and the fasting blood glucose levels of the OVX group,the DM group,and the O+D group were higher than those of the sham operation group.However,treatment with FTS·B and 17β-estradiol significantly reversed this trend.Compared with the Sham group,the fasting body weight in the DM group was significantly lower,and the fasting body weight in the OVX group was significantly higher.Compared with the O+D group,fasting body weight increased significantly in the OVX group at week 4,6,and 8,and the DM group decreased significantly at week 8.However,there were no significant differences between groups treated with different doses of FTS·B and17β-estradiol.Compared with Sham group,FBI levels in DM group and O+D group were significantly decreased.After treatment,FBI levels in ER,L-F and H-F groups were significantly improved.This suggests that FTS·B has an anti-diabetic effect.2 Behavioral experiment results2.1 Water maze experiment results: the water maze was used to assess the learning and memory of mice.Compared with Sham group,OVX,DM,and O+D groups showed more chaotic motion,longer escape delay,and reduced number of platform crossings.Among them,group O+D has the most obvious change.However,treatment with FTS·B and 17β-estradiol significantly reversed this trend,especially in the H-F group.There was no significant difference in swimming speed between the groups.Compared with Sham group,the target quadrant time in OVX group,DM group and O+D group was significantly shortened.Among them,group O+D was the most obvious.However,treatment with FTS·B and 17β-estradiol significantly reversed this trend,especially in the H-F group.2.2 Y Maze experiment results: Working memory and spatial memory performance were assessed by recording spontaneous changes in the Y-maze test.The results showed that there was statistical significance in spatial memory performance among all groups(P<0.05).Compared with Sham group,OVX group,DM group and O+D group had less spontaneous alternations.Among them,the spontaneous alternating motion in group O+D was the least.However,treatment with FTS·B and 17β-estradiol effectively increased the percentage of spontaneous changes.2.3 Results of new object recognition: In order to confirm the influence of FTS·B on menopausal diabetic encephalopathy,we also conducted new object recognition.Statistical analysis showed that compared with Sham group,OVX,DM and O+D groups showed significantly reduced interest in objects during the test phase,especially in new objects.However,treatment with FTS·B and17β-estradiol significantly reversed this trend.3 Histological test resultsWe evaluated whether FTS·B treatment affected hippocampal morphology in each group of mice.We found that the hippocampal morphology of the Sham group was normal,with obvious nucleoli and clear nuclear membranes.OVX group,DM group and O+D group had loss of hippocampal neurons,sparse arrangement of hippocampal cells and blurred boundaries.The O+D group was the most severely affected.Importantly,treatment with FTS·B and 17β-estradiol largely reversed these abnormalities,especially in the H-F group.These results suggest that FTS·B has a significant neuroprotective effect,as it inhibits the loss and structural changes of hippocampal neurons.4 Immunohistochemical staining resultsImmunohistochemical results showed that the number of Aβ-positive plaques in the hippocampus of OVX group,DM group and O+D group was significantly higher than that of Sham group,and the difference was most significant in O+D group.However,intervention with FTS·B and17β-estradiol significantly reduced Aβ deposition in group O+D,especially in group H-F.In this study,hyperaggregation of p-tau was found in the hippocampus of mice in the OVX,DM and O+D groups compared with the Sham group.p-tau superaggregation was most severe in group O+D.These differences were significantly reduced after treatment with FTS·B and17β-estradiol,especially in the H-F group.5 Results of Real-time PCR and Western blot5.1 Detection of synaptic factors: We detected the expression levels of synapse-associated proteins(PSD-95,synaptophysin,and synaptophysin-1)in the hippocampus,which constitute the molecular basis of synaptic plasticity.Compared with Sham group,the levels of PSD-95,synaptogenin and synapsin-1 in OVX,DM and O+D groups were significantly decreased.The decrease of group O+D was the most obvious.After treatment with FTS·B and17β-estradiol,the expressions of these three histones were significantly increased in ovariectomized diabetic mice,especially in the H-F group.The results of RT-PCR were consistent with those of western blot.These results suggest that FTS·B enhances the expression level of synapse-related proteins in the hippocampus,which may indicate that FTS·B has a dose-dependent effect in vivo.5.2 Detection of inflammatory factors: We studied whether neuroinflammation exists in postmenopausal diabetic patients and whether FTS·B can prevent neuroinflammation.RT-PCR and western blot were used to detect the expression levels of TNF-α,IL-6 and IL-1β in the hippocampus.RT-PCR results showed that compared with Sham group,the expression levels of TNF-α,IL-6 and IL-1β in the hippocampus of OVX group,DM group and O+D group were significantly increased,and in the O+D group.FTS·B and17β-estradiol significantly reduced the m RNA expression levels of these factors,and this effect was most pronounced in the HF group.The results of western blot were consistent with those of RT-PCR(P<0.05).These results indicated that FTS·B inhibited hippocampal neuroinflammation in ovariectomized diabetic mice.Conclusions:1.Oophorectomy can successfully simulate postmenopausal diabetes in diabetic mice,providing animal model basis for subsequent treatment and intervention.2.Estrogen deficiency is one of the risk factors for exacerbating cognitive degeneration in postmenopausal diabetic mice.Early intervention with FTS·B can improve the cognitive function of ovariectomized diabetic mice.3.FThe potential mechanism of FTS·B alleviating cognitive impairment in ovariectomized diabetic mice may be by inhibiting neuroinflammation and improving synaptic plasticity.