叉分蓼总黄酮调控铁死亡治疗帕金森病的作用机制OA

OBJECTIVE To investigate the mechanism through which total flavonoids of Polygonum divaricatum L.(TFP)regulate ferroptosis for the treatment of Parkinson's disease(PD).METHODS① Core ferroptosis-related differential genes involved in TFP's treatment of PD were identified via bioin-formatics analysis and molecular docking technology.② Animal experiments:C57BL/6 mice were randomly divided into the control group,the model group,the model+TFP 0.78 g·kg-1 group,and the model+TFP 1.56 g·kg-1 group.Mice in the model+TFP groups received corresponding treatment once daily by intragastric administration for 14 consecutive days.Except the control group,PD was induced in each group by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)(30 mg·kg-1)on experimental days 5 to 9.Pole climbing and balance beam tests were used to evaluate the motor coordination ability of PD mice.After completion of behavioral tests,immunohistochemistry was performed to detect the expression levels of tyrosine hydroxylase(TH),α-synuclein(α-syn),ionized calcium-binding adaptor molecule-1(IBA-1),and glial fibrillary acidic protein(GFAP)in the substantia nigra-striatum(SN-str)of mice.The levels of total iron(Fe)and ferrous iron(Fe2+)in brain tissue were determined by the ferrozine colorimetric assay.The content of malondialdehyde(MDA)was measured using the thiobarbi-turic acid(TBA)assay while the level of total-superoxide dismutase(T-SOD)was detected by the WST-1 assay.ELISA was used to measure the levels of Fe2+,glutathione synthetase(GSS),glutathione(GSH),and reactive oxygen species(ROS).RT-qPCR was conducted to detect the mRNA expressions of Gss,autophagy-related gene 7(Atg7),and acyl-CoA synthetase long-chain family member 4(Acsl4)in brain tissues.Western blotting was used to determine the protein expression levels of GSS,ATG7,ACSL4,GSH peroxidase 4(GPX4),lysophosphatidylcholine acyltransferase 3(LPCAT3),and nuclear receptor coactivator 4(NCOA4)in brain tissues.RESULTS Bioinformatics analysis and molecular docking confirmed that Gss,Atg7,and Acsl4 were the core targets of TFP in regulating ferroptosis in PD.Animal experiments showed that compared with the control group,there was a significant decrease in motor coordination,massive loss of TH-positive dopaminergic neurons in the substantia nigra-striatum,a surge in α-syn protein levels,and significant increases in the number and expression levels of IBA-1-and GFAP-positive cells accompanied by typical activation-related morphological alterations in the model group.At the mechanistic level,mRNA levels of Atg7 and Acsl4 were significantly elevated,Gss mRNA levels were significantly decreased in brain tissues,ferroptosis-related proteins ACSL4 and LPCAT3 as well as ferri-tinophagy pathway molecules ATG7 and NCOA4 were significantly upregulated,GPX4 protein expres-sion was significantly reduced,the contents of Fe,Fe2+,ROS and MDA in brain tissues were increased while the levels of GSH,GSS and T-SOD were significantly decreased in the model group.Compared with model group,the aforementioned pathological injuries and molecular abnormalities were effectively mitigated in that motor function was significantly restored,the loss of TH-positive neurons was attenuated,α-syn levels were reduced,the abnormal increases in IBA-1-and GFAP-positive cells were significantly inhibited,activation-related morphological alterations were markedly ameliorated,the mRNA levels of Atg7 and Acsl4 were decreased,Gss mRNA levels were significantly increased,the protein expression levels of ACSL4,LPCAT3,ATG7,and NCOA4 were significantly downregulated,the contents of Fe,Fe2+,ROS and MDA were significantly reduced,and the levels of GSH,GSS and T-SOD were significantly elevated in the model+TFP group.However,no significant difference was observed in GPX4 protein expressions.CONCLUSION TFP can effectively ameliorate both pathological damage and motor dysfunction in MPTP-induced PD mice.The underlying mechanism may involve the modulation of the ferroptosis pathway by targeting such core proteins as GSS,ATG7 and ACSL4.