Effect And Its Mechanism Of Dihydromyricetin On Intracerebral Hemorrhage Mice

Objective: The pathologic mechanisms of intracerebral hemorrhage(ICH)are still unknown,and access to treatment is limited,and the vast majority of patients receive extremely poor outcomes.Therefore,it is necessary to develop new drugs to change the current treatment predicament of ICH.Dihydromyricetin(DMY)has a variety of physiological functions and has been found to have a satisfactory effect in the treatment of intracerebral infarction.However,the role of DMY in ICH and its underlying mechanism remain unclear.The objective of this study was to investigate the effect and potential mechanism of DMY in the treatment of ICH mice.Methods: After confirming the beneficial effect of DMY on ICH mice,this study preliminarily determined that DMY may play a therapeutic role by inhibiting the expression of Lipocalin-2(LCN2)through genomics and network pharmacology.Subsequently,data from the National Center of Biotechnology Information(NCBI)database and Immunofluorescence(IF),Immunohistochemical(IHC),Western blot(WB),enzymerelated immunosorbent assay(ELISA)and quantitative real-time polymerase chain reaction(q RT-PCR)were used to evaluate the expression and protein levels of LCN2 gene in ICH models;Adenovirus overexpressing LCN2 was injected into the basal ganglia of C57BL/6J mice,treated with DMY and a series of response experiments were performed to determine the intervention effect of DMY on LCN2 and the role of LCN2 in ICH models.First,ferroptosis and iron deposition were detected by WB,iron staining and electron microscopy.Then,the downstream target of LCN2 was explored by proteomics.Finally,computational docking analysis,co-Immunoprecipitation(Co-IP)and molecular biology were used to verify the mechanism.Results: DMY can promote hematoma absorption in ICH mice,improve neurobehavioral abnormalities,and reduce brain tissue cell apoptosis and abnormal number of neurons.Transcriptomic and network pharmacology results suggest that LCN2 may be the molecular target of DMY therapy in ICH mice.The expression of LCN2 was increased in the mice brain tissues after ICH,and DMY inhibited the expression of LCN2.Subsequent response experiments confirmed that overexpression of LCN2 can reduce the therapeutic effect of DMY on ICH mice,indicating that LCN2 is the molecular target of DMY on ICH mice.LCN2 is a protein closely related to iron transport and ferroptosis.The effects of DMY/LCN2 on iron deposition and ferroptosis in ICH mouse brain tissue were evaluated.The results showed that the expressions of cyclooxygenase-2(COX2)and phosphate extracellular regulated protein kinase(p-ERK)were significantly decreased after DMY treatment.Ferroptosis decreased and the number of abnormal mitochondria decreased under electron microscopy after DMY treatment.This improvement was reversed after overexpression of LCN2.Then,proteomics found that Solute Carrier Family 3 Member 2(SLC3A2)may be the downstream target of LCN2 promoting ferroptosis.Finally,it was found that LCN2 strongly bonded with SLC3A2,a component of the cystine/glutamate reverse transport system(system Xc-).The synthesis of glutathione(GSH)and the expression of glutathione peroxidase-4(GPX4)were affected,thus promoting the process of ferroptosis,and DMY could reverse this process.This further validates the aforementioned conclusion.Conclusions: DMY can improve the symptoms of ICH mice by inhibiting LCN2 expression.The potential mechanism is that DMY reverses the inhibitory effect of LCN2 on systerm Xc-,thereby alleviating ferroptosis in brain tissue and exerting therapeutic effect.Part Ⅰ: DMY Improves Symptoms of ICH Mouse ModelObjective: The purpose of this section was to determine the therapeutic effect of DMY on ICH mice.Methods: ICH mouse model was constructed after 7 days of prophylactic DMY intragastric treatment,and continued intragastric treatment was performed for 3 days after modeling.Some mice were continued to be treated until the behavioral evaluation time point,so as to evaluate the therapeutic effect of DMY on ICH mice The experimental groups were:(1)Sham+vehicle group,(2)ICH+vehicle group,(3)ICH+DMY group.3 days after ICH modeling,the mice were sacrificed for hematoma measurement,and the brain tissue was made into paraffin sections.Terminal deoxynucleoitidyl transfrase-mediated d UTP nick end labeling(Tunel)staining and nissl staining were used to evaluate the apoptosis of neuronal cells and neurons in ICH mice.According to the corresponding time points,behavioral scores were performed on the mice 1 day before ICH modeling and 1,3,5,7 and 14 days after ICH modeling to evaluate the neurological impairment of the mice.Results: DMY can promote the absorption of hematoma in ICH mice,promote the recovery of neurological disability in ICH mice,and reduce the apoptosis of neuronal cells and neurons in the brain tissue around the hematoma in ICH mice.Conclusions: DMY can improve the symptoms of ICH mice and play a beneficial role in the treatment of ICH.Part Ⅱ: Molecular Target of DMY to Improve ICH Neural FunctionObjective: The first part of the experiment confirmed that DMY can improve the symptoms of ICH mice.The purpose of this part of the experiment was to find the molecular target of DMY in the treatment of ICH mice.Methods: ICH mouse model was established after 7 days of DMY intragastric treatment.After ICH modeling,the treatment was continued for 3 days,and the brain tissue was taken for genomic sequencing,so as to explore the molecular targets of DMY therapy for ICH mice.The experimental groups were:(1)Sham+vehicle group,(2)Sham+DMY group,(3)ICH+vehicle group,(4)ICH+DMY group.Genomic methods were used to search for differential expression genes(DEGs)after ICH in mice and treatment with DMY.Therapeutic targets of ICH mice treated with DMY were determined by network pharmacology.Molecular targets of ICH mice treated with DMY were determined by intersection of DEGs and therapeutic targets.Results: The results of genomic data analysis showed that there were 24 DEGs of ICH and after DMY treatment,of which 14 were down-regulated genes after ICH and up-regulated after DMY treatment,and 10 were up-regulated genes after ICH and down-regulated after DMY treatment.A total of 178 possible targets of DMY in the treatment of ICH mice were identified.The intersection of 24 DEGs and 178 therapeutic targets indicated that LCN2 may be the molecular target of DMY therapy for ICH mice.Conclusions: LCN2 may be the molecular target of DMY therapy in ICH mice.Part ⅡI: DMY Improved the Symptoms of ICH Mice by Inhibiting LCN2 ExpressionObjective: The above experimental results indicate that DMY may play a role in the treatment of ICH by regulating LCN2.This part of the experiment verifies the expression changes of LCN2 after ICH in mice,the influence of DMY on the expression level of LCN2 and the effect of LCN2 on the treatment effect of DMY on ICH mice.Methods: In the first step,ICH mouse model was constructed after 7 days of DMY intragastric treatment,and continued for 3 days after modeling.The experimental groups were:(1)Sham+vehicle group,(2)Sham+DMY group,(3)ICH+vehicle group,(4)ICH+DMY group.Firstly,the NCBI database was searched to analyze the expression changes of LCN2 after ICH in mice.Then the mice were killed 3 days after ICH modeling,and the brain tissue was made into paraffin sections.Part of the brain tissue was treated and stored for later use.Finally,the expression of LCN2 in brain tissue and serum of ICH mice was evaluated by IF,IHC,WB,q RT-PCR and ELISA.In the second step,after 3 days of DMY intragastric treatment,adenovirus overexpressing LCN2 was injected into the basal ganglia of mice,and then continued to use DMY intragastric treatment for 4 days to construct ICH mouse model.The mice were treated for 3 days after modeling,and some mice were treated until the time point of behavioral evaluation.The experimental groups were:(1)ICH+DMY+PBS group,(2)ICH+DMY+OE-Ctrl group,(3)ICH+DMY+OELCN2 group.3 days after ICH modeling,the mice were sacrificed for hematoma measurement,and WB and q RT-PCR were performed to evaluate the expression level of LCN2 after adenovirus transfection.At the same time,the brain tissue was made into paraffin sections,and the apoptosis of neuronal cells and neurons in the brain tissue of ICH mice was evaluated by Tunel staining and nissl staining.According to corresponding time points,behavioral scores were performed on mice 1 day before ICH and 1,3,5,7 and 14 days after ICH modeling to evaluate the neurological impairment of mice.Results: NCBI database results showed that LCN2 expression increased in ICH mice.After ICH in mice,LCN2 gene level and protein level were increased.DMY could reduce the expression of LCN2 in ICH mice.Adenovirus transfection significantly increased the expression of LCN2 in ICH mice and LCN2 overexpression could reduce the therapeutic effect of DMY on ICH mice.Conclusions: DMY can inhibit the expression of LCN2 after ICH in mice,and overexpression of LCN2 can partially reverse the therapeutic effect of DMY on ICH mice.Part IV: DMY may inhibit ferroptosis in ICH mice by interfering the binding of LCN2 to system Xc-Objective: In this part,the downstream molecular mechanism of DMY/LCN2 therapy in ICH mice was investigated.Methods: After 3 days of DMY intragastric treatment,adenovirus overexpressing LCN2 was injected into the basal ganglia of mice,and then continued to be given DMY intragastric treatment for 4 days to construct ICH mouse model.The mice were treated for 3 days after modeling,and some mice were treated for 14 days.The experimental groups were:(1)Sham+vehicle group,(2)ICH+vehicle group,(3)ICH+DMY+PBS group,(4)ICH+DMY+OE-Ctrl group,(5)ICH+DMY+OE-LCN2 group.Firstly,the changes of ferroptosis related proteins in brain tissue of mice in each group were detected by WB.At 3 and 14 days after ICH modeling,the mice were sacrificed,and the brain tissue was paraffin sliced for iron staining to evaluate iron deposition in ICH mice brain tissue.The microscopic morphology of ferroptosis in ICH mice was observed by electron microscope.Finally,proteomics,molecular docking and co-IP methods were used to find the downstream molecular pathway of DMY/LCN2 to improve ferroptosis in ICH mice,and related proteins were detected and verified.Results: DMY/LCN2 can improve iron deposition,reduce ferroptosis,reduce ferroptosis pathway related protein levels and reactive oxygen species(ROS)levels in ICH mice.systerm Xc-is the downstream molecular pathway of DMY/LCN2.DMY treated ICH mice by inhibiting the binding of LCN2 to systerm Xc-.Conclusions: DMY plays a therapeutic role by inhibiting the binding of LCN2 to systerm Xc-and alleviating ferroptosis in ICH mice.