| Objective To investigate whether the protective effects of total flavones of Dracocephalum moldavica L(TFD).on cerebral ischemia/reperfusion injury and hypoxia-reoxygenation injury of SH-SY5 Y cells and its mechanism are related to apoptosis signal pathway.Methods In vivo experiments,SD rats were used as research objects,and a model of middle cerebral artery embolism was established to explore the protective effects of different concentrations of TFD on cerebral ischemia/reperfusion injury in rats.Animal experiments: 60 SD rats were random Ly divided into a sham operation group(Sham),a model group(MCAO alone),TFD low-dose group(Low),intermediate-dose group(Intermediate),and high-dose group(High).(The administration group was orally administered with the TFD 25mg/kg/d,50mg/kg/d,100mg/kg/d for 7 days,and the sham operation group and the model group were administered with saline.)A total of 5 groups,12 in each group.In the sham operation group,only the blood vessels were separated,and no thread plug was inserted.The MCAO animal model was made by the model group and the drug treatment group according to the Zea-longa method.After 2 hours of ischemia,the plug was pulled out to cause reperfusion injury.After 22 hours,conduct behavioral testing for neurological deficits,and the serum inflammatory factor IL-6 and TNF-α,HE and Nissl staining to detect the pathological morphology of nerve cells,immunohistochemical staining and western blot to detect the expression of Caspase-3,Bax and Bcl-2.Real-time PCR was used to detect the expression of Caspase-3,Bax and Bcl-2 m RNA.In vitro experiments,a hypoxia/reoxygenation(H/R)injury cell model was established with SH-SY5 Y cells as the research object to clarify its specific mechanism.First,the degree of damage of sy5 y cells to hypoxia/reoxygenation at different times was determined to determine the maximum time point of cell damage.Cells were treated with different concentrations of TFD to study the optimal TFD drug concentration.The experimental cells were divided into 5groups: Control group(Control),model group(H/R),administration group(TFD+H/R),inhibitor group(LY294002+H/R),administration group + inhibitor group(TFD+LY294002+H/R).The contents of oxidative stress MDA,SOD,and CAT in the supernatant were detected by Elisa method;the expression of Bax and Bcl-2 in the cells was detected by immunofluorescence staining.The expression levels of Bax,Bcl-2 and PI3K/Akt protein in upstream signaling pathway were detected by western blot.Real-time PCR was used to detect Bax and Bcl-2 m RNA expression.Results(1)Compared with the sham group,the behavioral scores of the model group were significantly increased and statistically significant(P<0.05).The behavioral scores of the middle-dose and high-dose groups were decreased compared with the model group.And there was significant statistical significance(P<0.05,there was no significant difference in behavior scores between the low-dose group and the model group(P> 0.05).(2)Compared with the sham group,the serum inflammatory factor IL-6 and TNF-α are increased and have significant statistical significance(P<0.05).Compared with the model group,the serum inflammatory factors IL-6 and TNF-α are reduced and statistically significant compared with the model group(P<0.05).There was no significant difference in serum inflammatory factors IL-6 and TNF-α between the low-dose group and the model group(P>0.05).(3)Compared with the sham operation group,Nerve cells and vertebral body damage increased.Compared with the model group,the damage of nerve cells and vertebral body cells was reduced in the middle and high dose groups,while the damage of nerve cells and vertebral body cells was not reduced in the low dose group compared with the model group.(4)Compared with the sham operation group,the Caspase-3 and Bax proteins in the brain tissue of the model group Compared with the model group,the expression of Caspase-3 and Bax protein in the brain tissue decreased while the expression of Bcl-2protein decreased and the expression of Bcl-2 protein decreased significantly(P<0.05).The expression of Bcl-2 protein increased and had significant statistical significance(P<0.05).Compared with the model group,the expression of Caspase-3 and Bax proteins in the brain tissue increased and had significant statistical significance(P<0.05).But the expression of Bcl-2 protein was not statistically significant(P>0.05).(5)Compared with the sham group,the expression of Caspase-3 and Bax m RNA in the brain tissue increased and the expression of Bcl-2 m RNA decreased with statistical significance(P<0.05).Compared with the model group,the expression of Caspase-3 and Bax m RNA in the brain tissue was reduced and the expression of Bcl-2 m RNA was increased with statistical significance(P<0.05).Compared with the model group,There was no significant difference in the expression of Caspase-3,Bax,Bcl-2 m RNA in brain tissues(P> 0.05).(6)Compared with 2h of hypoxia,the cell viability was decreased and statistically significant(P <0.05).TFD at 5μg/m L could significantly protect nerve cell damage.(7)Compared with the control group,the content of oxidative stress factor MDA increased while the content of SOD and CAT decreased and the statistical significance was significant in the model group and inhibitor group(P<0.05).Compared with the model group,the content of oxidative stress factor MDA decreased while the content of SOD and CAT increased in the inhibitor group compared with the model group(P<0.05).(8)Compared with the control group,the expression of Bcl-2 protein in the model group and the inhibitor group was decreased while the expression of Bax protein was increased with significant statistical significance(P<0.05).Compared with the model group,the expression of Bcl-2 protein was increased while the expression of Bax protein was decreased in the inhibitor group compared with the model group(P <0.05).(9)Compared with the control group,the expression of Bcl-2 m RNA in the model group and the inhibitor group was decreased while the expression of Bax m RNA was increased with significant statistical significance(P<0.05).Compared with the model group,the expression of Bcl-2 m RNA increased and the expression of Bax m RNA decreased in the inhibitor group(P<0.05).(10)Compared with the control group,the expressions of p-PI3 K and p-Akt in the model group and inhibitor group were reduced and statistically significant(P<0.05).Compared with the model group,the The expressions of p-PI3 K and p-Akt were increased and had significant statistical significance(P<0.05).Compared with the administration group and the model group,p-PI3 K and p-Akt in the inhibitor group were significantly reduced and statistically significant.(P<0.05).Compared with the inhibitor group,p-PI3 K and p-Akt in the administration group + inhibitor group were significantly increased and statistically significant(P<0.05).Conclusions(1)total flavones of Dracocephalum moldavica L can improve the behavioral defects after cerebral ischemia-reperfusion model.(2)The total flavones of Dracocephalum moldavica L can reduce the content of IL-6 and TNF-ɑ inflammatory factors to achieve anti-inflammatory effect.(3)The total flavones of Dracocephalum moldavica L can reduce the expression of apoptosis-related factors Caspase-3 and Bax while increasing the expression of Bcl-2.(4)The total flavones of Dracocephalum moldavica L can regulate the content of oxidative stress molecules MDA,SOD and CAT and protect sy5 y cells induced by hypoxia-reoxygenation injury.(5)The total flavones of Dracocephalum moldavica L can regulate the expression of downstream apoptotic factors Bcl-2 and Bax by activating PI3 K / Akt signaling pathway. |
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