| Objective:To explore the effect of Chinese medicine Xijiao Dihuang Decoction on the level of autophagy in rats with focal cerebral ischemia reperfusion,and to study its specific regulation mechanism,to provide experimental basis and pharmacological basis for the clinical treatment of acute cerebral infarction.Methods:Through the preparation of middle cerebral artery occlusion(MCAO)in the middle cerebral artery,reperfusion was achieved 2 hours after modeling.The modeled rats were randomly divided into 6 groups according to random number method:Sham group,Model group,Xijiao Dihuang Decoction low dose group(XJDH-L group),Xijiao Dihuang Decoction high dose Group(XJDH-H group),Rapamycin group,Rhodiola sinensis high dose+rapamycin group(XJDH-H+Rapamycin group).The neurological deficit scores and cerebral infarction volume ratio were observed at different time points.The ultrastructure and autophagosome changes of nerve cells were observed by transmission electron microscopy.The pathological changes of brain tissue were observed by HE staining and immunofluorescence.Apoptosis was observed by TUNEL staining.Western blotting was used to detect the expression of autophagy-related proteins such as LC3-Ⅱ,Atg-5 and p62.Result:1.Effect of Xijiao Dihuang Decoction on Neurobehavioral of MCAO RatsAfter successful model establishment,at the time of reperfusion at Id,3d,and 7d,the neurobehavior scores were performed according to the Longa5 scoring method by observers who were unclear.The neurological function score of the sham-operated rats was 0,and there was no significant change in behavior.Rats in the MCAO model group showed significant neuromotor dysfunction,and the scores were significantly different(p=0.000).In the other groups,the neurological function scores were not significantly different from the model group on the first postoperative day(P>0.05).The rats in each group treated with Xijiao Dihuang Decoction had different degrees of decline on the 3rd and 7th day after operation compared with the first day(all P<0.05 or P<0.001),and the high dose group was more effective..At the same time,the neurological function score of rats in the high dose group of Xijiao Dihuang Decoction was lower than that in the MCAO model group(P<0.05).The results showed that Xijiao Dihuang Decoction can improve the neurobehavioral disorders of rats in different degrees,and the effect of high dose group is more obvious.2.Effect of Xijiao Dihuang Decoction on Brain Pathology of MCAO RatsAfter HE staining,it can be observed by light microscopy:the morphology of the brain tissue of rats in the sham operation group is regular,the arrangement is relatively neat,the nucleolus is clearly visible,and the nucleus is not deeply stained and condensed.In the model group,the cell structure of the rat brain was severely changed,showing vacuolization,nucleus lysing and deflation,and neuronal loss,which occurred in the central area of cerebral ischemia.No obvious neuronal loss was observed in the peripheral cortex.The cell structure destruction and the loss of ischemic neurons in the rapamycin group were more obvious than those in the model group.In contrast,the nerve cells in the low-dose and high-dose groups of Xijiao Dihuang Decoction showed mild ischemic changes,and no obvious cell swelling and necrosis and neuronal loss were observed.The results showed that Xijiao Dihuang Decoction has obvious improvement effect on nerve cell ischemia,and the effect of high dose of Xijiao Dihuang Decoction is more obvious.3.Effect of Xijiao Dihuang Decoction on infarct volume of MCAO ratsMRI revealed that there was no infarction in the brain tissue of the sham operation group.Cerebral ischemia was evident in the MCAO model group,and there was a significant difference in the scores(p=0.000).In addition to the rapamycin group,the rats in each group treated with Xijiao Dihuang Decoction had different degrees of cerebral infarction volume on the 3rd and 7th day after surgery(all P<0.05 or P<0.001).The results suggest that rapamycin aggravates the volume of cerebral infarction,and dif’ferent doses of Xijiao Dihuang Decoction can reduce the volume of cerebral ischemia to a certain extent,and the effect of high dose group is better.4.Effect of Xijiao Dihuang Decoction on the ultrastructure of autophagy in MCAO ratsElectron microscopy showed that the organelles in the neurons of the sham-operated rats were abundant,and there were few lysosomes,the mitochondria structure was clear,and the chromatin distribution was normal.In the model group and the rapamycin group,the hippocampal neurons showed large damage,the cells showed more vacuoles,the mitochondria broke,the lysosomes increased,and the autophagosome of the bilayer membrane structure was observed,and the undegraded organelle was contained inside,(mitochondria).Compared with the model group,the ultrastructure of the nuclear membrane of the neuron was more clear and complete,and the mitochondria in the cells were slightly swollen and a few layers of autophagosomes were observed.Compared with the rapa group,the rapamycin combined with Xijiao Dihuang Decoction soup was significantly reduced in autophagosomes,but the number of autophagosomes was higher than that in the high-dose group.The results suggest that the brain protection mechanism of Xijiao Dihuang Decoction may be related to the reduction of autophagosome formation.5.Effect of Xijiao Dihuang Decoction on activation and localization of autophagy inMCAO ratsUsing immunofluorescence double labeling method,it can be clearly seen under the microscope that LC-3Ⅱ and Beclin-1 in the model group showed green fl uorescence,NeuN was red fluorescence,and the ischemic penumbra was orange.The fluorescence intensity of LC-3Ⅱand NeuN was the highest,and the fluorescence intensity of LC-3Ⅱ and GFAP was weak.The results indicated that LC-3Ⅱ was mainly distributed in neuronal cells,while microglia and glial cells were rare.6.Effect of Xijiao Dihuang Decoction on Brain Cell Apoptosis in MCAO RatsThe apoptosis of TUNEL cells showed that the number of apoptotic cells(green fluorescent cells)in the group of Rhizoma Dihuang Decoction was significantly lower than that in the MCAO model group,further indicating that Xijiao Dihuang Decoction can alleviate neuronal cell damage and has neuroprotective effects.The number of apoptotic cells in the rapamycin group was significantly higher than that in the model group,indicating that rapamycin further aggravated brain damage by inducing autophagy.Compared with the high-dose group of Xijiao Dihuang Decoction,the number of apoptotic cells in the rapamycin combined with Xijiao Dihuang Decoction group increased significantly,indicating that rapamycin attenuated the neuroprotective effect of Dijiao Dihuang Decoction,indirectly indicating the anti-brain of Dijiao Dihuang Decoction Ischemia is closely related to autophagy.7.The expression of autophagy-related proteins LC3,Atg5 and p62 in MCAO rats and the molecular mechanism of autophagyThe experimental results showed that the model group and rapamycin LC3-11,Atg5 protein expression increased,LC3-Ⅱ/LC3-1 ratio increased,p62 expression decreased,autophagy level increased.The Xijiao Dihuang Decoction administration group can down-regulate the expression of LC3-Ⅱ and Atg5 protein and increase the level of p62 protein.The above results indicate that Xijiao Dihuang Decoction can inhibit the transformation of LC3-Ⅰ to LC3-Ⅱ and P62 protein degradation in ischemic penumbra cortex,thereby reducing the production of autophagosomes,reducing the level of autophagy and exerting neuroprotective effects;After the treatment of autophagy inducer Rapamycin,the opposite result was obtained,which further aggravated the degree of cerebral ischemia.Combined with Xijiao Dihuang Decoction,the neuroprotective effect of Xijiao Dihuang Decoction Maxim.PI3K,mTOR and ERK are upstream proteins of LC and are closely related to autophagy regulation.In the model group,the levels of mTOR and PI3K were decreased,and the phosphorylation level of ERK was increased.Xijiao Dihuang Decoction can promote the expression of mTOR and PI3K and reduce ERK phosphorylation.The results indicate that Xijiao Dihuang Decoction may inhibit ERK phosphorylation by promoting AKT-mTOR signaling pathway,thereby inhibiting autophagy of cerebral ischemia and improving cerebral ischemia-reperfusion inj ury.Conclusion:Xijiao Dihuang Decoction can improve the neurobehavioral of rats,reduce cell swelling and necrosis and neuronal loss,reduce the volume of cerebral infarction,reduce the formation of autophagosomes,and at the same time exert the mechanism of neuroprolection and promote the AKT-mTOR signaling pathway.Inhibition of ERK phosphorylation inhibits autophagy. |
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