| Objective:1 This study aimed to explore the protective impact of Buyang Huanwu Decoction(BHD)on a diabetic brain injury model in mice.2 Employing untargeted metabolomics techniques,our research examined the influence of Buyang Huanwu Decoction on the serum metabolic profiles in mice suffering from diabetic brain injuries,offering a novel perspective for assessing BHD’s therapeutic effectiveness in managing diabetic brain injuries.3 Based on cellular and animal experiments,we clarified the molecular mechani sm of Buyang Huanwu Decoction in treating diabetic brain injury by regulating the PGC-1α/MFN2/Drp-1 signalling axis,and provided a new strategy for Buyang Huan wu Decoction in treating diabetic brain injury.Methods:Experiment Ⅰ:Sixty c57BL/6J mice were randomly divided into six groups,including blank control group(Control),model group(Model),metformin group(Metformin),Buyang Huanwu Decoction high(BHD-H),medium(BHD-M),and low-dose group(BHD-L).control group was given normal chow,and the remaining five groups were given to construct a diabetic brain injury The remaining 5 groups were given to construct an animal model of diabetic brain injury with STZ(100mg/kg)combined with high-fat feed,and were given medication after the model was successfully constructed,in which the Buyang Huanwu Decoction high-dose,medium-dose,and low-dose treatment groups were converted to the equivalent dosage by adult body weight of 70 kg,and were gavaged once a day with different BHD decoctions according to 86.5g/kg,43.2g/kg,and 21.6g/kg,respectively.The Metformin group was administered at a dose of 130 mg/kg once a day;Control group and Model group were given saline 10 m L/kg,once daily by gavage for 10 weeks.After 10 weeks of drug administration,samples were taken,and HE staining was used to observe the pathological and morphological changes in the hippocampus of mice,ELISA detected Aβ amyloid in mouse brain tissue,and Western blot detected the expression of p-Tau(S396),p-Tau(S404)protein in brain tissue.Experiment Ⅱ:Modelling,grouping and drug administration methods were the same as in Experiment I.LC-MS technology was used to detect small molecule metabolites in serum before and after treatment in positive and negative ion mode,and metabolites were quantified,screened and annotated using software such as meta X and databases such as HMDB and KEGG.After that,Metaboanalyst 5.0 software and Microbiology Letter platform were applied to perform multivariate statistical analysis on the obtained 3D data matrix.Finally,according to the screening conditions,the differential metabolites among the three screened groups were analysed for pathway enrichment and their biological significance was clarified.Experiment Ⅲ:1 Cellular experiments: HT22 cells were induced by high sugar(100m M)(48h)to construct an in vitro model of diabetic brain injury,and the CCK-8 method was applied to detect the cytotoxicity of the Buyang Huanwu Decoction and its effect on the viability of HT22 cells induced by high sugar(100m M)as a means of determining the dose of BHD administered in the cellular experiments and determining the experimental grouping;the blank control group(Control),High sugar(100m M)group(Model),high dose(BHD-H),medium dose(BHD-M),low dose(BHD-L)amount group of tonic yang and five tonics group,and metformin group(Metformin),ELISA detected the content of ATP and ROS in the cells;Western blot and RT-PCR detected the content of PGC-1α/MFN2/Drp-1 signalling axis and the expression of mTOR,HIF-1α related genes and proteins in cells by Western blot and RT-PCR,respectively.2 In the animal study,the methodology for modeling replicated that of Experiment I.The content of leucine(Leu)in the mouse brain tissue was quantified using the Enzyme-Linked Immunosorbent Assay(ELISA).Furthermore,the expression levels of genes and proteins associated with the PGC-1α/MFN2/Drp-1 signaling axis,as well as those related to mTOR and HIF-1α,were examined in the brain tissue using Western blot and reverse transcription-polymerase chain reaction(RT-PCR)techniques,respectively.These methods were employed to investigate the molecular effects and potential therapeutic mechanisms of the interventions under study in the context of diabetic brain injury.Results:Experiment Ⅰ:(1)Fasting blood glucose: When compared to the Control group,the glucose levels in the bloodstream of the mice from the Model group saw a notable rise(P<0.01).Following a treatment period of 10 weeks,the glucose measurements in the BHD group’s mice demonstrated a significant downward trend(P<0.01).(2)HE staining: In Control group,the neuronal cells in the hippocampus of mi ce were closely arranged,with intact morphology,round and oval nuclei,uniform co louring,and no nuclear condensation or lysis.Compared with the Control group,thecells in the hippocampus of the Model group were scattered and disorganised,withwidened interneuron gaps,altered cell morphology,wrinkled cytosol,and deeply stai ned nuclei,and the cells of the BHD three groups and the Metformin treatment gro up were more densely packed and neatly arranged,with most of the neuronal cells r elatively intact,and with lighter alterations in cell morphology.(3)ELISA: In comparison to the Control group,the levels of Aβ in the brain tissue of the mice from the Model group exhibited a notable elevation(P<0.01);when contrasted with the Model group,the concentrations of Aβ in the brain tissue of mice within the BHD-H and BHD-M groups were observed to be on a decline,with this difference reaching statistical significance(P<0.05).(4)Western blot: Relative to the Control group,the expression levels of p-Tau(S396)protein were markedly elevated in the Model group(P<0.01),as were the levels of p-Tau(S404)protein(P<0.05).Following treatment with Tonifying Yang Returning Five Soup,all three BHD groups showed a significant reduction in p-Tau(S396)expression compared to the Model group(P<0.01).Additionally,p-Tau(S404)expression was reduced in both the BHD-H and BHD-L groups when compared with the Model group(P<0.05).Experiment Ⅱ:(1)On the basis of clarifying that there were metabolic differences between the groups,differential metabolites were screened from the results obtained,(screening conditions:(1)ratio≥2 or ratio≤1/2;(2)p-value <0.05;(3)VIP≥1),the metabolites obtained were screened,and 313 differential metabolites were finally obtained between the Model group and Control group,and 313 differential metabolites were obtained between the BHD group and the Model group,and 25 metabolites of difference between BHD group and Model group.After taking the intersection of the two,it was found that the metabolites abnormally raised or lowered in the Model group,and the metabolites that were regulated back after BHD treatment were 18 in total.(2)Differential metabolites of each group were imported into metaboanalyst 5.0and online analysis software of Microbiotics platform for enrichment analysis of met abolic pathways.deoxocucurbitacin I,arecoline,all-trans-retinoyl-β-glucuronide,19(r)-hydroxy-prostaglandin e2,19(r)-hydroxy-prostaglandin e2,and all-trans-retinoyl-β-glucu ronide.prostaglandin e2,riboflavin tetrabutyrate,epitestosterone;4-methylphenol,dieth ylmaleate,isoliquiritigenin(isoliquiritigenin).isoliquiritigenin,evoxanthidine,metazachl or,maltol,dibenzothiophene,and thymol sulfate.18 differential metabolites were mai nly enriched in valine,leucine,and isoleucine biomass.leucine and isoleucine biosyn thesis,retinol metabolism,valine,leucine and isoleucine degradation,flavonoid biosy nthesis and other metabolic pathways.Experiment Ⅲ:Cell experiments:(1)Proliferative activity: Following a 48-hour exposure to a high glucose concentration of 100 m M,the viability of HT22 cells was reduced to 48%,showing a significant deviation from the results observed in both the Control group and the mannitol group(P<0.01).(2)Administration concentration: Concentration of Administration: At 48 hours post-treatment with BHD on HT22 cells,the peak in cellular activity was noted at a concentration of 200 μg/m L(P<0.05).Further,upon BHD treatment of HG-HT22 cells for48 hours,a notable enhancement in the viability of HG-HT22 cells was observed at concentrations of 200,400,and 600 μg/m L(P<0.01).Additionally,the application of metformin at concentrations of 2 and 4 m M significantly boosted the viability of HG-HT22cells(P<0.01).(3)ELISA:The ATP concentration was markedly reduced in the HT22 cells of the Model group when contrasted with the Control group(P<0.01).Post-treatment,the ATP concentrations witnessed a significant increase in the BHD-M and BHD-L groups relative to the Model group(P<0.01).Regarding the ROS levels,HT22 cells in the Model group exhibited a substantial increase compared to those in the Control group(P<0.01).Following treatment,the ROS levels significantly decreased in the treated cells in comparison to the Model group(P<0.01).(4)PCR:mTOR mRNA expression: The level of mTOR mRNA expression in the Model group showed a significant reduction when compared to the Control group(P<0.01).After undergoing treatment,there was a significant increase in gene expression within the BHD-H,BHD-L,and Metformin groups compared to the Model group(P<0.01).HIF-1α mRNA expression: The expression level of HIF-1α mRNA was higher in the Model group than in the Control group,with the difference being statistically significant(P<0.05).Following treatment,there was a significant reduction in gene expression levels across all treatment groups when compared to the Model group(P<0.01).PGC-1α mRNA expression: PGC-1α mRNA expression was found to be lower in the Model group in comparison to the Control group,with the variance being statistically significant(P<0.05).Post-treatment,there was a significant increase in the gene expression levels in the groups that received administration compared to the Model group(P<0.01).MFN2 mRNA expression: MFN2 mRNA expression was reduced in the Model group when compared with the Control group(P<0.05).Following the treatment,gene expression levels in the treatment groups were up-regulated compared to the Model group,with the BHD-H,BHD-L,and Metformin groups experiencing more significant up-regulation(P<0.01),and the BHD-M group showing a trend towards increased expression(P<0.05).Drp-1 mRNA expression: Drp-1 mRNA expression was significantly elevated in the Model group in comparison to the Control group(P<0.01).Following treatment,the BHD-H,BHD-M,and Metformin groups showed a significant decrease in expression(P<0.01),and the BHD-L group also exhibited a significant reduction in comparison to the Model group,with the differences being statistically significant(P<0.05).(5)Western blot:mTOR protein expression was significantly reduced in the Model group when compared to the Control group(P<0.01).Following treatment,protein expression levels in all the treated groups were higher than in the Model group,with the BHD-L group exhibiting a more pronounced increase in protein expression(P<0.01).HIF-1α protein expression was elevated in the Model group compared to the Control group(P<0.05).Post-treatment,there was a reduction in protein expression across all four administered groups relative to the Model group,with the Metformin and BHD-L groups showing a more notable trend towards down-regulation(P<0.01).The BHD-H and BHD-M groups also experienced a statistically significant down-regulation in comparison to the Model group(P<0.05).PGC-1α protein expression: PGC-1α protein expression in the Model group was significantly reduced compared to the Control group(P<0.05).Following treatment,the protein expression in the BHD-L group was significantly elevated compared to the Model group(P<0.05).MFN2 protein expression: MFN2 protein expression was significantly decreased in the Model group in comparison to the Control group(P<0.05).Following treatment,the Metformin and BHD-L groups showed a statistically significant recovery in protein expression compared to the Model group(P<0.05).Drp-1 protein expression: Drp-1 protein expression was significantly elevated in the Model group compared to the Control group(P<0.05).Following treatment,both the Metformin and BHD-L groups exhibited a significant decrease in expression,which was statistically significant in comparison to the Model group(P<0.05).Animal experiment:(1)ELISA: Leu level: When compared to the Control group,the level of Leu in the brain tissue of mice in the Model group was significantly decreased(P<0.01).Relative to the Model group,Leu levels in the brain tissues of mice in the Metformin,BHD-H,BHD-M,and BHD-L groups exhibited a tendency to revert towards normalcy,with the changes being statistically significant(P<0.05).(2)PCR:mTOR mRNA expression: The mTOR mRNA expression level was significantly lower in the Model group compared to the Control group(P<0.01).Following treatment,gene expression in the four treated groups was elevated to varying extents compared with the Model group,with a statistically significant increase observed in the BHD-H and BHD-L groups(P<0.01),and a notable up-regulation in the Metformin and BHD-M groups(P<0.05).HIF-1α mRNA expression: The expression level of HIF-1α mRNA was higher in the Model group than in the Control group,with this difference being statistically significant(P<0.05).After treatment,when compared to the Model group,all four administered groups exhibited varying degrees of down-regulation.The decrease in the BHD-M group was significant(P<0.01),and the down-regulation in the remaining three groups was also significant,with statistically significant differences(P<0.05).PGC-1α mRNA expression: Compared to the Control group,PGC-1α mRNA expression in the Model group was significantly lower(P<0.01).After treatment,relative to the Model group,all four administered groups saw an increase in expression to varying extents.The up-regulation was particularly notable in the BHD-M group(P<0.01),and the other three groups also experienced significant increases,with statistically significant differences(P<0.05).MFN2 mRNA expression: Compared to the Control group,the MFN2 mRNA expression in the Model group was significantly reduced,showing a statistically significant difference(P<0.05).After treatment,in comparison to the Model group,all four administered groups experienced an increase in expression to varying degrees.The Metformin,BHD-M,and BHD-L groups showed a more significant upregulation(P<0.01),while the BHD-H group also displayed significant upregulation,with a statistically significant difference(P<0.05).Drp-1 mRNA expression: Drp-1 mRNA expression was elevated in the Model group compared to the Control group,with a statistically significant difference(P<0.05).Following treatment,all four administered groups showed a decrease in expression when compared to the Model group,with a more pronounced downward trend observed in the Metformin and BHD-H groups(P<0.01).The BHD-L group also experienced significant down-regulation,with this difference being statistically significant(P<0.05).(3)Western blot:For mTOR expression,the protein levels in the Model group were lower compared to those in the Control group,with this difference being statistically significant(P<0.05).Post-treatment,protein expression in the BHD-M and BHD-L groups saw an increase compared to the Model group(P<0.05),with the Metformin group exhibiting a more pronounced increase(P<0.01).Regarding HIF-1α expression,protein levels were higher in the Model group compared to the Control group,marking a statistically significant difference(P<0.05).After treatment,all three BHD groups showed lower protein expression levels than the Model group.The decrease was more marked in the BHD-L group(P<0.01),with the BHD-H and BHD-M groups also experiencing statistically significant reductions in protein expression(P<0.05).or PGC-1α expression,the Model group exhibited significantly lower PGC-1α protein levels compared to the Control group(P<0.05).Post-treatment,there was an increase in protein expression in the BHD-M group relative to the Model group(P<0.05),with a more pronounced elevation observed in the BHD-L group(P<0.01).In terms of MFN2 expression,the Model group showed decreased MFN2 protein levels compared to the Control group(P<0.05).Following treatment,protein expression was significantly upregulated in both the BHD-H and BHD-L groups compared to the Model group,marking a statistically significant change(P<0.05).Drp-1 expression saw an increase in the Model group when compared with the Control group,showing a statistically significant difference(P<0.05).Following treatment,protein expression in all four administered groups decreased to varying extents compared to the Model group.The BHD-H and Metformin groups experienced a statistically significant reduction in protein expression(P<0.05),while the decrease in the BHD-M and BHD-L groups was even more significant(P<0.01).Conclusion:(1)Buyang Huanwu Decoction has been shown to lower blood glucose levels,enhance the neuronal condition in the hippocampus of mice with diabetic brain injury,and decrease the levels of Aβ amyloid as well as the expression of p-Tau(s396)/p-Tau(s404)proteins in the brain tissues.These actions contribute to mitigating the impacts of diabetic brain injury.(2)By modulating the serum metabolic profile and stabilizing glucose and lipid metabolism in mice with diabetic brain injury,Buyang Huanwu Decoction aids in the improvement of diabetic brain injury.(3)Buyang Huanwu Decoction protects neuronal mitochondrial structure,encourages mitochondrial regeneration,and maintains mitochondrial fission/fusion homeostasis by modulating the PGC-1α/MFN2/Drp-1 signaling axis and the expression of mTOR and HIF-1α genes and proteins.This multifaceted approach helps to mitigate the damage to HG-HT22 cells and diabetic brain injury in mice with diabetes. |
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