Observation Of The Effect Of Zhilong Huoxue Tongyu Capsule On Rats With Ischemic Stroke And The Effect Of Neuronal Mitochondria Based On Energy Metabolism

Objective:By establishing a rat model of acute ischemic stroke,giving different doses of Zhilong Huoxue Tongyu Capsules to observe the effect of Zhilong Huoxue Tongyu Capsules on rats with ischemic stroke,and explore its effects on energy metabolism and brain The influence of cortical neuron mitochondrial structure and function.Provide reference and ideas for the clinical application of Zhilong Huoxue Tongyu Capsule and the next experimental research.Methods:1.Modeling and grouping:120 clean-grade male SD rats were selected.Except for the sham-operated control group,all the others were used to prepare the rat middle cerebral artery occlusion（MCAO）model by the modified suture method.Evaluation was performed 24 hours after the operation.Successful models were randomly included in the model control group,ZL 0.25g/kg group,ZL 0.5 g/kg group,ZL 1 g/kg group,and citicoline sodium tablet group 0.0625 g/kg group,20 per group.The drugs in each administration group were prepared with 5%sugar saline.The sham operation control group and the model control group were given the same volume of 5%sugar saline for 14consecutive days.2.Observation of various indicators:（1）General status;（2）Neural function score;（3）TTC staining to observe the ischemic side infarct volume;（4）HE staining to observe the pathological morphology of brain tissue;（5）Enzymatic chemistry method to detect serum energy metabolism related parameters（glucose,lactic acid）,Pyruvate);（6）Enzymatic chemistry method to detect cerebral cortex energy metabolism related parameters（glucose,lactate,pyruvate）;（7）Enzymatic chemistry method to detect cerebral cortex mitochondrial ATP content and ATPase activity(Na⁺-k⁺-ATP,Ca²⁺-Mg²⁺-ATP);（8）Transmission electron microscopy to observe the mitochondrial ultrastructure of cerebral cortex neurons on the ischemic side;（9）Flow cytometry to detect the mitochondrial membrane potential of cerebral cortex neurons（JC-1method）.Results:1.General status:The rats in the sham-operated group are obese,have smooth fur,have good spirits and activities,and have no abnormalities in diet and feces.The rats in the model group were thin and weak,with yellow and dry hair,slow response,little activity,little or no food,and some rats had hematuria and loose stools.Compared with the model group,the ZL 0.5,1 g/kg dose group had different degrees of recovery in spirit,activity,diet,etc.after the 3rd to 4th day of administration.Weight change:Weigh all the rats on the14th day after operation and compare them.Compared with the model group,the weight of rats in each group increased significantly（P<0.01）.2.Neurological function score:Neurological function score:All rats in the sham operation group had no neurological deficit symptoms during the entire experimental period,while the neurological deficit symptoms of rats in the model group were very obvious,and the scores continued to be high.Compared with the model group,the neurological function scores of the drug groups on the 7th and 14th days after the operation were reduced to different degrees.Among them,the ZL 0.5 and 1 g/kg dose groups were significantly reduced at the two time points（P<0.05 or P<0.01）.3.Brain tissue infarct volume:Compared with the sham operation group,the infarct volume ratio of the model group was significantly higher（P<0.01）,indicating that the model was successful.Compared with the model group,the infarct volume ratio in the ZL0.5 and 1 g/kg dose groups decreased（P<0.01）,and the ZL 0.25 g/kg dose group had a decreasing trend（P<0.05）.4.Histopathological morphology of cerebral cortex:the sham operation group has complete cortical structure,with more neurons and neatly arranged.In the model group,the cortical structure is loose,the number of neurons is very small,and the cells have obvious pathological changes.Compared with the model group,the pathological changes of each dose group of ZL were reduced to varying degrees.5.The effect of ZL on the related parameters of serum energy metabolism in MACO rats:（1）Serum glucose content:Compared with the sham operation group,the serum glucose content of rats in the model group was significantly reduced（P<0.01）.Compared with the model group,the serum glucose content of each drug group was increased（P<0.01）,among which ZL was dose-dependent among each group.（2）Serum lactic acid content:Compared with the sham operation group,the serum lactic acid content of the model group increased significantly.Compared with the model group,the serum lactic acid content in each drug group was reduced to varying degrees,among which the ZL 0.5 and1 g/kg dose groups had significant statistical significance（P<0.01）.（3）Serum pyruvate content:Compared with the sham operation group,the serum pyruvate content of rats in the model group decreased（P<0.01）.Compared with the model group,the serum pyruvate content of each drug group recovered to varying degrees,and the difference of ZL 0.25 g/kg was statistically significant（P<0.05）.The difference between the 0.5 and 1 g/kg dose groups was statistically significant（P<0.01）.（4）Serum lactate/pyruvate ratio:Compared with the sham operation group,the serum lactate/pyruvate ratio of rats in the model group was significantly increased（P<0.01）.Compared with the model group,the serum lactic acid/pyruvate ratio of each drug group decreased to varying degrees,and the difference was significant（P<0.01）.6.Parameters related to energy metabolism of cerebral cortex:（1）Glucose content of cerebral cortex:Compared with the sham operation group,the cerebral cortex glucose content of rats in the model group was significantly reduced（P<0.01）.Compared with the model group,the brain cortex glucose content of each dose group of ZL increased to different degrees（P<0.05 or P<0.01）.（2）The content of lactic acid in the cerebral cortex:Compared with the sham operation group,the content of lactic acid in the cerebral cortex of the model group increased significantly（P<0.01）.Compared with the model group,the content of lactic acid in the cerebral cortex of each dose group of ZL decreased（P<0.05 or P<0.01）.（3）The content of pyruvate in the cerebral cortex:Compared with the sham operation group,the content of pyruvate in the cerebral cortex of rats in the model group was significantly reduced（P<0.01）.Compared with the model group,the content of pyruvate in the cerebral cortex of the ZL 0.5 g/kg dose group increased（P<0.05）,and the content of ZL 1 g/kg dose increased significantly（P<0.01）.（4）The ratio of lactic acid/pyruvate in the cerebral cortex:Compared with the sham operation group,the ratio of lactic acid/pyruvate in the cerebral cortex of the model group was significantly increased（P<0.01）.Compared with the model group,the ratio of lactic acid/pyruvic acid in the cerebral cortex of the ZL 0.5 and 1 g/kg dose groups decreased to varying degrees,and the differences were statistically significant（P<0.01）.7.Mitochondrial ATP content and ATPase activity of cerebral cortex neurons:（1）The mitochondrial ATP content of cerebral cortex neurons:Compared with the sham operation group,the mitochondrial ATP content of neurons in the model group decreased significantly（P<0.01）.Compared with the model group,there was no significant increase in neuronal mitochondrial ATP content in the ZL 0.25 g/kg dose group（P>0.05）;the ZL 0.5 and 1 g/kg dose groups both increased significantly（P<0.01）.（2）The activities of mitochondrial Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase of cerebral cortex neurons:Compared with the sham operation group,the activities of mitochondrial Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase of neurons in the model group were significantly decreased（P<0.01）.Compared with the model group,mitochondrial Na⁺-K⁺-ATPase activity and Ca²⁺-Mg²⁺-ATPase activity in ZL0.5 and 1 g/kg dose groups were significantly increased（P<0.01）.However,there was no statistically significant increase in the ZL 0.25 g/kg dose group（P>0.05）.8.The ultrastructure of cerebral cortex neuron mitochondria:The mitochondrial morphology of cerebral cortex neuron in the sham operation group was prolonged,and the membrane and cristae structure were normal.The shape of mitochondria in the model group was mainly round,the cristae dissolved and broke,or even disappeared,showing a vacuole-like change.After treatment with drugs,mitochondrial damage was reduced to varying degrees.Compared with the model group,the number of mitochondria in the ZL0.5 and 1 g/kg dose groups increased,the membrane structure became clear,and the cristae structure damage was reduced.The larger the dose,the smaller the damage.9.Mitochondrial membrane potential of cerebral cortex neurons:Compared with the sham operation group,most of the mitochondrial membrane potential of the model control group decreased.Among them,the proportion of mitochondria with normal membrane potential decreased significantly,while the proportion of mitochondria with decreased membrane potential Significantly higher,with significant statistical difference（P<0.01）.Compared with the model control group,the ratio of mitochondria with normal membrane potential in each dose group of ZL increased,while the proportion of mitochondria with decreased membrane potential decreased to varying degrees,and the differences were statistically significant（P<0.01）.Conclusion:1.Zhilong Huoxue Tongyu Capsules can improve the neurological deficit symptoms and brain tissue pathological damage in rats with ischemic stroke.2.Zhilong Huoxue Tongyu Capsules can effectively regulate the energy metabolism of blood and brain cells in rats with ischemic stroke.3.Zhilong Huoxue Tongyu Capsule can reduce the general structure and function damage of cortical neuron mitochondria in rats with ischemic stroke,and has a good neuroprotective effect.