Study On The Mechanism Of Didangtang Regulating Mitochondrial Autophagy And Improving Nerve Damage After Cerebral Hemorrhage Based On The Nrf2/ARE Signal Pathway

Objective:This experiment is divided into animal experiments and cell experiments.It is based on the Nrf2/ARE signal transduction pathway.Through the study of mitochondrial autophagy,it explains the mechanism of resistance decoction to treat cerebral hemorrhage,and clarifies that resistance decoction relieves hematoma in rats with cerebral hemorrhage.The intervention mechanism of surrounding tissue damage provides a more reliable experimental basis for the clinical application of this prescription in the treatment of acute cerebral hemorrhage.Methods:Establish a rat autologous blood infusion model of cerebral hemorrhage and a CoCl₂-induced hypoxia model of PC12 cells,and randomly divide them into a sham operation group(control group),model group,Naoxuekang control group,low,medium,and high-dose levels of Didangtang group;Longa scoring method was used to observe the effect of Didang decoction in improving neurological deficits in rats with cerebral hemorrhage;Tunel staining was used to detect the effect of Didang decoction on cell apoptosis in focal tissues of cerebral hemorrhage rats;flow cytometry was used to detect the effect of Didang decoction on CoCl₂-induced PC12 cell apoptosis;Western blot was used to detect the influence of Didang Decoction on PC12 cell apoptosis-related proteins induced by cerebral hemorrhage in rats and CoCl₂to determine the neuroprotective effect of Didang Decoction.qPCR method was used to detect the effect of Didang decoction on the copy number of mitochondrial DNA(mtDNA)in intracerebral hemorrhage in rats'brain tissue and PC12 cells induced by CoCl₂;fluorescence method was used to detect the effect of Didang decoction on the ATP content of brain tissue in rats with intracerebral hemorrhage and PC12 cells induced by CoCl₂;Biochemical method was used to detect the influence of Didangtang on SOD,CAT,GSH-px,MDA in brain tissue of rats with cerebral hemorrhage;fluorescence labeling method was used to detect the influence of Didangtang on the mitochondrial membrane potential of PC12 cells induced by CoCl₂.Western blot method was used to detect the effect of Didang Decoction on the expression of autophagy-related proteins in the brain tissue of rats with intracerebral hemorrhage and CoCl₂-induced PC12cells;immunohistochemical method was used to detect the effect of Didang Tang on the autophagy-related proteins in brain tissue of rats with intracerebral hemorrhage.Western blot was used to detect the influence of Didang Decoction and Nrf2 inhibitor on the Nrf2/ARE signal pathway of PC12 cells induced by CoCl₂;flow cytometry was used to detect the effect of Didang Decoction and Nrf2 inhibitor on the apoptosis of PC12 cells induced by CoCl₂;the qPCR method was used to detect effect of Didang Decoction and Nrf2 inhibitor on mtDNA of PC12 cells induced by CoCl₂.Fluorescence method was used to detect the effect of Didang Decoction and Nrf2 inhibitor on ATP content in CoCl₂-induced PC12 cells;Flow cytometry was used to detect the effect of Didang Decoction and Nrf2 inhibitor on the influence of cell mt ROS in CoCl₂-induced PC12.Results:(1)Didang Decoction can improve the neurological function score of rats with cerebral hemorrhage and promote the recovery of neurological function after cerebral hemorrhage.(2)Didang Decoction can down-regulate the expression levels of neuronal apoptosis-related proteins Bax and Caspase-3 and increase the expression level of Bcl-2 in cerebral hemorrhage rats and CoCl₂-induced PC12 cell hypoxia model,thereby reducing neuronal apoptosis.(3)In the rat cerebral hemorrhage model and the CoCl₂-induced PC12 cell model,it was found that Didangtang can reduce mtDNA copy number,increase ATP content,and restore mitochondrial membrane potential,indicating that Didangtang can improve the mitochondria of damaged nerve cells after cerebral hemorrhage.(4)In the rat model of cerebral hemorrhage,it was found that Didang Decoction can increase the content and activity of SOD,CAT,GSH-px and inhibit the content of MDA,indicating that Didang Decoction can inhibit the oxidative stress of damaged nerve cells after cerebral hemorrhage.(5)In the rat cerebral hemorrhage model and the CoCl₂-induced PC12 cell model,it was found that Didangtang can increase the expression of Parkin,Pink1 and LC3II,inhibit the expression of P62 protein,and promote mitochondrial autophagy.(6)Didang Decoction induces mitochondrial autophagy by activating the Nrf2/ARE pathway,thereby improving mitochondrial function and alleviating the neurological damage induced by hypoxia.Conclusion:Experiments have confirmed that Didang Decoction can significantly improve the neurological function score of cerebral hemorrhage model rats and inhibit neuronal apoptosis;experiments have confirmed that its protective effect is through regulating the Nrf2/ARE signaling pathway,promoting mitochondrial autophagy,thereby reducing mtDNA copy number and increasing ATP content,restore mitochondrial membrane potential,inhibit oxidative stress and improve mitochondrial function,thereby exerting neuroprotective effects.