| Objective:Based on the similarities between the theory of Qi deficiency and blood stasis in TCM and mitochondrial function,a successful MCAO rat model of Qi deficiency and blood stasis syndrome was made.Observe the improvement of Buyang Huanwu Decoction on the four diagnosis and brain tissue morphology of model rats,as well as the degree of recovery of mitochondrial function and the regulation of related proteins in the mitochondrial biosynthesis pathway.Explore the biological connotation of Qi deficiency and blood stasis syndrome in ischemic stroke and the mechanism of Buyang Huanwu Decoction.Methods:Forty healthy male clean SD rats were divided into blank group,model group,Buyang Huanwu Decoction group,and butyl phenyl peptide group by random number table method,with 10 rats in each group.The rats in the blank group were kept for two consecutive weeks without any treatment.The other three groups all adopted the traditional Chinese medicine"work causes Qi loss" and "hungry causes Qi loss" etiological modeling methods,simulating hunger factors and fatigue factors,and continuously intervened for 14 days to prepare rat models of Qi deficiency and blood stasis syndrome.On the 15th day,the MCAO rat model was prepared by the thread plug method,and finally an animal model combining disease and syndrome was formed.After the modeling was completed,the drugs were administered according to different groups,and the drugs were administered continuously for 14 days.According to the original prescription of Buyang Huanwu Decoction,the decoction-free granules were configured and converted into the daily amount of crude drug for rats according to the amount of crude drug required by adults.The value of Buyang Huanwu Decoction group was 12.825g/kg,and the butyl phenyl peptide group was 54mg/kg,blank The group and the model group were given equal volumes of normal saline.After the administration,the general state of the rats in each group was observed,and the holding power,exhausted swimming time,pulse frequency,and tongue RGB value of each group were measured.After that,the brain was decapitated by anesthesia,and the morphological changes of the brain tissue in the ischemic penumbra area were observed by HE staining;the mitochondrial respiratory chain enzyme complex Ⅰ-Ⅳ activity and ATP content in the ischemic penumbra area were detected by the biochemical test kit to determine the mitochondria Functional changes.Western blotting was used to observe the expression of AMPK,PGC-1a,NRF-1,NRF-2,and mtTFA related to the mitochondrial biogenesis pathway.Result:1.Four diagnosis changes of Qi deficiency and blood stasis syndrome in MCAO rats:Compared with the blank group,rats in the model group were sluggish,weak in body,slow to respond to external stimuli,slightly open eyes,unhappy,lethargic,loose stools,withered hair and paw.The nail color is purple and dark.When walking,the limbs are weak,or they cannot move forward,or paralyzed and cannot wals.Holding power,exhausted swimming time and pulse frequency were significantly reduced(P<0.01).Compared with the model group,the rats in the Buyang Huanwu Decoction group have improved their general state,lack of energy,and respond to external stimuli They occasionally lie still,reduce limb hemiplegia,improve physical weakness,loose stools,and slightly hair Luster,claw nail color dark red,grip and pulse frequency increased(P<0.01),but there was no difference in exhaustive swimming time(P>0.05).2.Morphological changes of brain tissue:The brain tissue structure of the blank group is normal,the neurons are arranged neatly,the structure is clear and complete,the cytoplasm is lightly stained and the nucleus is in the middle.In the model group,the brain tissue structure is disordered,the cells are arranged irregularly,the nuclei are pyknotic and deeply stained,some cells are degenerated and necrotic,the cytoplasm and nucleus are blurred,the nerve fibers are spongy,and a large number of vacuoles appear.In the Buyang Huanwu Decoction group,the arrangement of brain cells is sparse and disordered,the nucleus pyknosis and deep staining are reduced,the cell degeneration is reduced,and some cells can see clear boundaries between cytoplasm and nucleus,and reduced vacuoles.3.Results of the activity of mitochondrial respiratory chain complex Ⅰ-Ⅳ:Compared with the blank group,the activity of mitochondrial complex Ⅰ-Ⅳ in the model group decreased significantly(P<0.01);compared with the model group,the activity of mitochondrial complexⅠ-Ⅳ in the butyl phenyl peptide group had different degrees Increased(P<0.01,P<0.05);Compared with the model group,the activities of mitochondrial complex Ⅰ and complex Ⅳof Buyang Huanwu Decoction group increased(P<0.01),but complex Ⅱ and complex Ⅲwas no difference in the activity of(P>0.05).4.Results of ATP content detection in brain tissue:Compared with the blank group,the ATP content of rats in the model group was significantly decreased(P<0.01);compared with the model group,the ATP content of rats in the Buyang Huanwu Decoction group and the butyl phenyl peptide group was significantly increased(P<0.01);Compared with the Buyang Huanwu Decoction group,the ATP content of the butyl phenyl peptide group was higher than that of the Buyang Huanwu Decoction group(P<0.05).5.AMPK expression results:Compared with the blank group,the expression of AMPK protein in the brain tissue of the model group was significantly increased(P<0.01);compared with the model group,the expression of AMPK protein in the brain tissue of the Buyang Huanwu Decoction group and the butyl phenyl peptide group was significantly decreased(P<0.01).Compared with the Buyang Huanwu Decoction group,the expression of AMPK protein in the brain tissue of the butyl phenyl peptide group was lower than that of the Buyang Huanwu Decoction group(P<0.01).6.PGC-1a,NRF-1,NRF-2,mtTFA expression results:There was no significant difference in the expression of PGC-1a in the brain tissue of rats in each group(P>0.05).Compared with the blank group,the expressions of NRF-1,NRF-2,and mtTFA in the brain tissue of the model group were significantly decreased(P<0.01,P<0.05);compared with the model group,the Buyang Huanwu Decoction group and the butyl phenyl peptide group were larger.The expression of NRF-1 in rat brain tissue was significantly increased(P<0.01);compared with the Buyang Huanwu Decoction group,the expression of NRF-1 in the brain tissue of rats in the butyl phenyl peptide group was higher than that in the Buyang Huanwu Decoction group(P<0.01).Conclusion:1.Establish and evaluate the combined disease and syndrome animal model of Qi deficiency and blood stasis syndrome in ischemic stroke through the idea of cause-syndromedisease-treatment.Observe the rat’s general state,holding power,exhausted swimming time,pulse frequency,and tongue RGB value to reflect the syndrome characteristics of Qideficiency and blood stasis,and observe the pathological changes of brain tissue to reflect the nature of the disease.Using Buyang Huanwu Decoction is effective for the treatment of the model to disprove the success of the model2.Through the intervention of Buyang Huanwu Decoction,the symptoms of the fourth diagnosis of Qi deficiency and blood stasis syndrome in MCAO rats have been improved,but when the rats are overworked,the body’s endurance is still in a state of insufficient,which shows the production and recovery of Qi It takes a relatively long process,so it is inferred that increasing the time of intervention will be more conducive to the improvement of symptoms.3.In ischemic stroke,the function of brain mitochondrial conplex Ⅰ-Ⅳ of Qideficiency and blood stasis syndrome is impaired,and the production of ATP is reduced.AMPK is overactivated.The activation of AMPK can continue to induce the expression of PGC-1a,but PGC-1a may fail to regulate NRF-1 and NRF-2,which in turn hinders the expression of mtTFA.The above protein expression disorder may be one of the pathways that affect the biogenesis of mitochondria,hinder the expression of mitochondrial respiratory chain complex proteins,and cause mitochondrial dysfunction.4.When Buyang Huanwu Decoction intervenes,AMPK expression decreases;PGC-1a is activated,and the activated PGC-1a may regulate the expression of downstream factor NRF-1.However,the expression of NRF-2 is not obvious,and the expression of mtTFA is regulated by NRF-1 and NRF-2,so the expression of mtTFA is not ideal.Therefore,the mechanism of Buyang Huanwu Decoction may be shown to improve the expression of AMPK/PGC-1a/NRF1 pathway proteins,promote the biogenesis of mitochondria,and thus may promote the activity of mitochondrial complex Ⅰ and complex Ⅳ,and increase the generation of ATP.It is one of the pathways to protect the function of mitochondria.5.Although Buyang Huanwu Decoction does not significantly improve the expression of proteins in the mitochondrial biogenesis pathway,it has a sufficient regulatory effect on AMPK.Therefore,it is speculated that Buyang Huanwu Decoction has a certain effect on other pathways of AMPK-mediated mitochondrial quality control. |
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