| Ischemic stroke is a common and frequently-occurring disease that seriously endangers human health.Cerebral ischemia/reperfusion(I/R)injury is an important pathophysiological process of ischemic stroke,which seriously affects the clinical treatment and prognosis of ischemic stroke.Autophagy is the main mechanism of maintaining cellular metabolic balance by degrading intracellular harmful proteins and damaged organelles,and plays an important role in the progression and prognosis of cerebral I/R injury.AMPK/ULK1 signaling pathway can sense intracellular energy changes and is a key pathway to regulate autophagy initiation.Buyang Huanwu Decoction(BHD)is a classic prescription of Traditional Chinese Medicine for the treatment of stroke caused by Qi deficiency and blood stasis.BHD still has significant effects on both the acute and convalesce stage of ischemic stroke at present.Buyang Huanwu Decoction has the effect of beneficial Qi,invigorating blood and clearing collaterals.It can replenish Yang,make Qi flourish and promote blood circulation,and restore the balance of Qi and blood as well as the balance of Yin and Yang in the body.It is similar to autophagy in maintaining the balance of cell metabolism.However,it is still unclear whether autophagy plays an important role in the inhibition of cerebral ischemia/reperfusion injury by BHD,and whether AMPK/ULK1 signaling pathway is involved in.Therefore,this study explored whether BHD alleviated cerebral I/R injury by regulating autophagy in vivo and in vitro,and furtherly explored whether the AMPK/ULK1 signaling pathway was involved in the regulation of autophagy by BHD in vitro.In this study,adult Sprague Dawlay(SD)rats and primary rat neurons were used to establish models to simulate the process of cerebral I/R injury,and the research was divided into three parts: 1.Effect of BHD on alleviation of cerebral ischemia/reperfusion injury.2.Study on the effect of BHD on alleviating cerebral ischemia/reperfusion injury by inhibiting autophagy.3.Study on the effect of BHD on alleviating cerebral ischemia/reperfusion injury by regulating autophagy through AMPK/ULK1 signaling pathway.Part one The effect of BHD on alleviating cerebral ischemia/reperfusion injury.Objective: To explore the alleviating effect of BHD on cerebral I/R injury,and to select the best action conditions.Methods: Adult SD rats were randomly divided into Sham group,I/R group and I/R+BHD group.The middle cerebral artery embolization/reperfusion(MCAO/R)model was used to simulate the brain I/R injury.After 2 h of ischemia,reperfusion was performed.At 1 d,3 d and 7 d after reperfusion,the brains of rats were taken.TTC staining was used to detect the cerebral infarction volume,TUNEL staining was used to detect the apoptosis rate,and Nissl’s staining was used to observe the neuronal injury.To explore the optimal action conditions of BHD for alleviating MCAO/R injury in rats.The rat cortical neurons were extracted and cultured for 7 days.Firstly,the conditions for the establishment of the OGD/R model of neurons were explored.The neurons were randomly divided into Control group and OGD/R group.Neurons in OGD/R group were subjected to oxygen and glucose deprivation for 1 h,2 h,3 h,4 h and reoxygenation for 24 h,respectively.The survival rate of neurons was measured by CCK-8 method and the release rate of LDH was measured by LDH method to determine the conditions for the establishment of neuron OGD/R model.Secondly,the neurons were randomly divided into Control group,OGD/R group,Buyang Huanwu Decoction Serum(BHDS)group(1%,3%,5%,10%)and CS group(1%,3%,5%,10%)based on the determined OGD/R model.The survival rate of neurons was detected by CCK-8 method,and the optimal concentration of BHDS was explored.Finally,the optimal concentration of BHDS was used to intervene OGD/R injured neurons.The morphological changes of neurons were observed by inverted microscope and the apoptosis rate of neurons was detected by flow cytometry to further clarify the protective effect of BHDS on OGD/R neurons.Results:1.Researches in vivo:1.1 Effects of BHD on the volume of cerebral infarction in rats: no infarction lesion was found in the brain tissues of the Sham group.The left cerebral tissue of I/R group was significantly infarct on day 1,3,and 7(P<0.01).Compared with I/R group,the cerebral infarct volume of rats in I/R+ BHD group was significantly reduced at 3 d(P<0.01)and 7 d(P<0.05)after reperfusion and treatment of BHD.The difference between the I/R group and the I/R+ BHD group for 3 d after reperfusion was slightly greater than that for 7 d.1.2 Effects of BHD on apoptosis of nerve cells in rat brain tissue: Almost no apoptosis was observed in the Sham group.Compared with the Sham group,the apoptosis rate of nerve cells in the left brain tissue of the I/R group at 1 d,3 d,and 7 d was significantly increased(P<0.01).After 3 d and 7 d intragastric administration of BHD,the apoptosis rate of nerve cells in the left brain of I/R+ BHD group was significantly decreased(P<0.01).The difference between the I/R group and the I/R+ BHD group after reperfusion for 3 d was slightly greater than that for 7 d.1.3 Effects of BHD on brain neuron injury in rats: cortex neurons in the Sham group had complete morphology,large cell body,deep staining of Nissl body and damaged rate of nerve cells was low.Compared with the Sham group,the cortical neurons in the left brain tissue of the I/R group were shrunk or even died at 1 d,3 d and 7 d after reperfusion,and the damaged rate of nerve cells was significantly increased(P<0.01).Compared with I/R group,the morphology of neurons in I/R+ BHD group was significantly improved after 3 d and 7 d of treatment of BHD,and the damaged rate of nerve cells was significantly decreased(P<0.01).The difference between the I/R group and the I/R+ BHD group after reperfusion for 3 d was slightly greater than 7 d.2.Researches in vitro:2.1 Cell observation and purity identification of primary cultured rat cortical neurons: The neurons were cultured 1-7 days after extraction and grew in good condition.On the 7th day,the neurons had full cell body and strong refractive activity,and abundant synaptic networks were formed between the cells.The purity of neurons was more than 95%.2.2 OGD/R model of neurons: compared with Control group,the survival rate of neurons with oxygen and glucose deprivation for 1 h,2 h,3 h,4 h,and reoxygenation for 24 h were significantly decreased(P<0.01),and the LDH leakage rates were significantly higher(P<0.01).With the increase of oxygen and sugar deprivation time,neuron survival rate gradually reduced,LDH leakage rate increased,rendering time dependence.The survival rate and LDH leakage rate of neurons were closest to the 50% inhibition rate when oxygen and glucose were deprived for 3 h and reoxygenated for 24 h.Therefore,the OGD/R model was established at this time point.2.3 Exploration of concentration of BHDS: compared with the Control group,the survival rate of neurons in OGD/R group was significantly decreased(P<0.01).After treatment with 1%BHDS(P<0.05),3%BHDS(P<0.01)and 3%CS(P<0.01),the survival rate of OGD/R neurons was significantly increased,and the survival rate of OGD/R neurons was significantly decreased after 5%BHDS,5%CS,10%BHDS and 10%CS intervention(P<0.01),but there was no significant change in the survival rate of neurons after 1%CS intervention.The survival rate of neurons in 3%BHDS group was significantly higher than that in 3%CS group(P<0.01),and the survival rate of neurons in 5%BHDS group was significantly higher than that in 5%CS group(P<0.01).Therefore,3%BHD serum was selected for the follow-up study.2.4 Protective effect of BHDS on OGD/R neurons2.4.1 Effects of BHDS on the morphology of OGD/R neurons: The neuron body in the Control group was full,and with good refraction,forming a uniform and dense neural network;Neurons in the OGD/R group were swollen or even disintegrated,and synapses were reduced and broken.After 3%BHDS intervention,the degree of cell body swelling of OGD/R neurons decreased,synapses increased significantly,and connections were formed between neurons.Cell body swelling was reduced,synapses was increased in the OGD/R neurons treated with 3% CS,connections was formed between individual neurons,but the improvement was not as significant as that in the 3%BHDS group.2.4.2 Effects of BHDS on the apoptosis rate of OGD/R neurons: compared with the Control group,the apoptosis rate of OGD/R group was significantly increased(P<0.01);After treatment of BHDS and CS,the apoptosis rate of OGD/R neurons was significantly decreased(P<0.01).The neuronal apoptosis rate in BHDS group was significantly lower than that in CS group(P<0.05).Summary: BHD can significantly reduce the cerebral infarction volume,the rate of nerve cell apoptosis,improve the injury of neurons,and alleviate the cerebral I/R injury in rats.Among them,cerebral ischemia for 2h and reperfusion for 3d,and treatment with BHD at the same time of reperfusion were the best conditions.BHDS can significantly increase the survival rate of OGD/R neurons,improve cell morphology,reduce cell apoptosis rate,and play a protective role in OGD/R neurons.Among them,3h of oxygen-glucose deprivation following by 24 h of reoxygenation and treatment with 3% BHDS were the best conditions of action.Part two The effect of BHD on inhibiting autophagy regulation for alleviating cerebral ischemia reperfusion injuryObjective: To explore the specific role of autophagy in brain I/R injury and whether BHD plays an inhibitory effect on brain I/R injury by regulating autophagy.Methods: SD rats were randomly divided into Sham group,I/R group and I/R+BHD group.MCAO/R model was established on rats of I/R group and I/R+BHD group after 2 h ischemia and 3 d reperfusion.I/R+ BHD group was given rats BHD(14.3g/kg)by intragastric gavage after reperfusion.The expression levels of Beclin-1 and LC3 B in the central,penumbra and contralateral regions of cerebral ischemia in rats were detected by Western Blot.The neuronal damage and autophagy in the penumbra region were observed by transmission electron microscopy.In vitro,neurons were randomly divided into Control group,OGD/R group,3-MA group(3mmol/L),Rapa group(500nmol/ L),BHDS group(3%),and BHDS+ Rapa group.OGD/R model was established for neurons after 3 h oxygen-glucose deprivation and 24 h oxygen-glucose reoxygenation.The expression of Beclin-1 and LC3 B in neurons was detected by Western Blot,the fluorescence intensity of CYTO-ID staining of neuronal autophagy was detected by flow cytometry,the fluorescence spot intensity of LC3 B and morphological changes of neuronal were detected by double immunofluorescence method,the survival rate of neurons was detected by CCK-8 method,and the apoptosis rate of neurons was detected by flow cytometry.Results:1.Researches in vivo:1.1 Effects of BHD on the levels of autophagy related proteins in different regions after cerebral I/R injury in rats: Beclin-1 expression in the central region of I/R group was significantly decreased compared with that in the Sham group(P<0.05),but there was no significant difference between the two groups.Transform from LC3B-Ⅰ to LC3B-Ⅱ had no significant difference in the three groups.Penumbra area I/R group of Beclin-1 expression and transform from LC3B-Ⅰ to LC3B-Ⅱ than Sham group increased significantly(P <0.01);compared with I/R group,Beclin-1 expression and transform from LC3B-Ⅰ to LC3B-Ⅱ in I/R+BHD group were significantly lower(P <0.05).The expression of Beclin-1 and transform from LC3B-Ⅰ to LC3B-Ⅱ had no significant difference between the three groups in the contralateral area1.2 Effects of BHD on cortex neurons damage and autophagy in the penumbra region of rats were observed by transmission electron microscopy: the neuron structure in the Sham group was intact,and no typical autophagy was found.In the I/R group,the neuronal membrane was damaged with severe edema,and more lipofuscin and autophagosomes were observed.The edema of neurons in I/R+ BHD group was improved,and the autophagy lysosomes were isolated.The degree of autophagy in I/R+ BHD group was lower than that in I/R group.2.Researches in vitro:2.1 Effect of BHDS on the expression of autophagy protein in OGD/R neurons2.2.1 Beclin-1 expression in each group: Beclin-1 level in OGD/R neurons was significantly higher than that in the Control group(P<0.01);Compared with OGD/R group,3-MA group(P<0.01)and BHDS group(P<0.05)were significantly decreased,but Rapa group was significantly increased(P<0.01);Beclin-1 level in BHDS+Rapa group was significantly lower than that of Rapa group(P<0.05),but still significantly higher than that of BHDS group(P<0.05).2.1.2 LC3 B expression in each group: after OGD/R injury of neurons,transform from LC3B-Ⅰ to LC3B-Ⅱ increased significantly(P < 0.01);compared with OGD/R group,3-MA group(P<0.05)and BHDS group(P<0.05)were significantly decreased,and Rapa group was significantly increased(P<0.05).BHDS+ Rapa group was significantly lower than that of Rapa group(P<0.01),but still significantly higher than that of BHDS group(P<0.05).2.2 Effects of BHD on the formation of autophagosomes in OGD/R neurons: compared with the Control group,the fluorescence signal intensity of CYTO-ID in OGD/R group was significantly increased(P<0.01);compared with OGD/R group,the fluorescence signal intensity of 3-MA group and BHDS group was significantly decreased(P<0.01),and that of Rapa group was significantly increased(P<0.01).The fluorescence intensity in BHDS+Rapa group was significantly decreased compared with that in Rapa group(P<0.01),but was still significantly higher than that in BHDS group(P<0.01).2.3 Effects of BHDS on LC3 B fluorescence spots in rat cortical neurons after OGD/R: LC3 B fluorescence intensity was low in neurons of the Control group,and synapses were abundant,forming neural networks.The fluorescence intensity of LC3 B in OGD/R injured neurons was significantly increased(P<0.01),the number of synapses was significantly decreased and the mean length was shortened.Compared with the OGD/R group,the fluorescence intensity of LC3 B in the 3-MA group(P<0.01)and the BHDS(P<0.05)group was significantly decreased,the morphology of neurons was improved,and the number of synapses was increased,and a small number of synaptic networks were formed between neurons in the BHDS group.The fluorescence intensity of LC3 B in Rapa group was significantly increased(P<0.01),and the number of synapses was decreased and shortened.The fluorescence intensity of LC3 B in BHDS+Rapa group was significantly lower than that in Rapa group(P<0.05),but it was still significantly higher than that in BHDS group(P<0.01),and the neuron morphology was slightly improved compared with that in Rapa group.2.4 Effects of BHDS on autophagy regulation on the survival of OGD/R neuron: compared with the Control group,the survival rate of OGD/R group was significantly decreased(P<0.01).Compared with the OGD/R group,the survival rate of neurons in the 3-MA group and the BHDS group was significantly increased(P<0.01),while that in the Rapa group was significantly decreased(P<0.01).The survival rate of neurons in BHDS+Rapa group was significantly lower than that in Rapa group(P<0.01),but still significantly higher than that in BHDS group(P<0.01).2.5 Effects of BHD on autophagy on OGD/R neuronal apoptosis: compared with the Control group,the apoptosis rate in OGD/R group was significantly increased(P<0.01).Compared with OGD/R group,the apoptosis rate in3-MA group and BHDS group was significantly decreased(P<0.01),but it was significantly increased in Rapa group(P<0.01).The neuronal apoptosis rate in BHDS+Rapa group was significantly lower than that in Rapa group(P<0.01),but still significantly higher than that in BHDS group(P<0.01).Summary: BHD can significantly reduce the expression of Beclin-1,conversion of LC3B-Ⅰ to LC3B-Ⅱ and the number and autophagosome,and alleviate cerebral I/R injury.BHDS can significantly reduce the expression of Beclin-1,conversion of LC3B-Ⅰ to LC3B-Ⅱ,the level of autophagosome and LC3 B fluorescence intensity of OGD/R injury neurons,improve the morphology of neurons,decrease the rate of neuronal apoptosis,improve the survival rate,and play a protective role for the OGD/R neurons.Part three The effect of BHD on alleviating cerebral ischemia reperfusion injury by inhibiting autophagy through AMPK/ULK1 signaling pathwayObjective: To explore the role of AMPK/ULK1 in OGD/R injury of neurons and whether BHD can inhibit cerebral I/R injury by regulating autophagy through AMPK/ULK1 signaling pathway.Methods: Neuronal OGD/R model was established,and the neurons were randomly divided into Control group,OGD/R group,Compound C group(2μmol/L),AICAR group(3mmol/L).The expression of p-AMPK,AMPK,p-ULK1,ULK1 and Beclin-1 in neurons was detected by western blot.The intensity and morphological changes of fluorescent spots in LC3 B neurons were detected by double immunoassay,and the survival rate of neurons was detected by CCK-8 assay.Neuronal OGD/R model was established,and the neurons were randomly divided into Control group,OGD/R group,AICAR group(3mmol/L),BHDS group(3%),and BHDS+AICAR group.The expression of p-AMPK,AMPK,p-ULK1,ULK1 and Beclin-1 in neurons was detected by western blot.The intensity and morphological changes of fluorescent spots in LC3 B neurons were detected by double immunoassay,and the survival rate of neurons was detected by CCK-8 assay.Results:1.The AMPK/ULK1 signaling pathway regulates autophagy and is involved in neuron OGD/R injury1.1 Regulation of the effect of AMPK on AMPK/ULK1 signaling pathway protein in OGD/R neurons: neuronal p-AMPK /AMPK was significantly increased after OGD/R injury(P<0.01).Compared with OGD/R group,p-AMPK/AMPK of neurons in Compound C group was significantly decreased(P<0.01),while that in AICAR group was significantly increased(P<0.01).Expression of p-ULK1 and ULK1 protein in neurons: p-ULK1/ULK1 in neurons after OGD/R injury was significantly increased(P<0.01).Compared with OGD/R group,p-ULK1 /ULK1 of neurons in Compound C group was significantly decreased(P<0.01),while that in AICAR group was significantly increased(P<0.05).1.2 Effects of AMPK regulation on Beclin-1 expression in OGD/R neurons: Beclin-1 protein expression in neurons: the level of Beclin-1 in neurons after OGD/R injury was significantly increased(P<0.01).Compared with OGD/R group,the level of Beclin-1 in Compound C group was significantly decreased(P<0.05),while that in AICAR group was significantly increased(P<0.05).1.3 Effects of AMPK regulation on LC3 B fluorescence spots and morphological changes of OGD/R neurons: LC3 B spots in the Control group had low fluorescence intensity and intact morphology.After OGD/R injury,the fluorescence intensity of LC3 B was significantly increased(P<0.01),and the number and length of synapses were significantly decreased.Compared with OGD/R group,LC3 B fluorescence intensity of neurons in Compound C group was significantly decreased(P<0.01),and the number of synapses was increased.The fluorescence intensity of LC3 B neurons in AICAR group was significantly increased(P<0.05),and the morphological damage was further aggravated.1.4 Effects of AMPK regulation on the survival rate of OGD/R neurons:the survival rate of neurons after OGD/R injury was significantly lower than that of Control group(P<0.01).Compound C group significantly increased the survival rate compared with OGD/R group(P<0.01).The survival rate of AICAR group was significantly lower than that of OGD/R group(P<0.01).2.BHDS regulates autophagy to alleviate cortical neuron OGD/R injury through AMPK/ULK1 signaling pathway2.1 Effects of BHDS on AMPK/ULK1 signaling pathway protein of OGD/R cortical neurons: AMPK activity of neurons after OGD/R injury was significantly increased(P<0.01).Compared with the OGD/R group,the neuronal AMPK activity of BHDS group was significantly decreased(P<0.05),and that of AICAR group was significantly increased(P<0.01).The neuronal AMPK activity in BHDS+AICAR group was significantly lower than that in AICAR group(P<0.01),but still significantly higher than that in BHDS group(P<0.05).The p-ULK1/ULK1 was significantly increased after OGD/R injury(P<0.01).Compared with OGD/R group,the p-ULK1/ULK1 in BHDS group was significantly decreased(P<0.01),and that in AICAR group was significantly increased(P<0.05).the p-ULK1/ULK1 in BHDS+AICAR group was significantly lower than that in AICAR group(P<0.01),but still significantly higher than that in BHDS group(P<0.05).2.2 Effects of BHDS on Beclin-1 expression after AMPK regulation in OGD/R neurons: Beclin-1 level was significantly increased after OGD/R injury(P<0.01).Compared with OGD/R group,the level of Beclin-1 in BHDS group was significantly decreased(P<0.05),while that in AICAR group was significantly increased(P<0.01).The level of Beclin-1 in BHDS+AICAR group was significantly lower than that in AICAR group(P<0.01),but still significantly higher than that in BHDS group(P<0.01).2.3 Effects of BHD on the fluorescence spots and morphological changes of OGD/R neurons under the regulation of AMPK: LC3 B in the Control group had low fluorescence intensity and intact morphology.After OGD/R injury,the fluorescence intensity of LC3 B was significantly increased(P<0.01),and the number and length of synapses were significantly decreased.The fluorescence intensity of LC3 B in AICAR group was significantly higher than that in OGD/R group(P<0.05),and the morphological damage of neurons was further aggravated.The fluorescence intensity of LC3 B in BHDS group was significantly lower than that in OGD/R group(P<0.01),and the number and length of synapses were improved.The fluorescence intensity of LC3 B in BHDS+AICAR group was significantly lower than that in AICAR group(P<0.01),and the morphology of the neurons was improved,but the fluorescence intensity of LC3 B was still significantly lower than that in BHDS group(P<0.01),and the synapses were not as rich as that in BHDS group.2.4 Effects of BHD on AMPK regulation on the survival rate of OGD/R neurons: the survival rate of neurons after OGD/R injury was significantly reduced(P<0.01).Compared with OGD/R group,the survival rate of neurons in BHDS group was significantly increased(P<0.01),and the survival rate of neurons in AICAR group was significantly decreased(P<0.01).The neuronal survival rate in BHDS+AICAR group was significantly higher than that in AICAR group,but still significantly lower than that in BHDS group(P<0.01).Summery: The activities of AMPK,ULK1 and Beclin-1 and the fluorescence intensity of LC3 B are significantly increased after OGD/R injury.Inhibition of AMPK activity can significantly reduce the activities of AMPK and ULK1,the level of Beclin-1 and the fluorescence intensity of LC3 B,and significantly improve the survival rate and morphology of neurons.Activating AMPK has the opposite effect.These results indicate that AMPK/ULK1 signaling pathway is activated after OGD/R and up-regulated autophagy level,which has a damaging effect on neurons.BHDS can significantly reduce the activity level of AMPK and ULK1,the level of Beclin-1,the fluorescence intensity of LC3 B,the survival rate and morphology of the OGD/R neurons.These results indicate that BHDS can inhibit the degree of hyperautophagy activation through AMPK/ULK1 signaling pathway,reduce the level of autophagy,and thus play a protective role on OGD/R neurons.Conclusion:1.BHD can reduce the cerebral infarction volume,the rate of nerve cell apoptosis,and improve the neuron damage in the brain tissue of MCAO/R rats.BHDS can increase the survival rate of OGD/R neurons,reduce the rate of apoptosis,improve the neuron morphology,and thus reduce the brain I/R injury.2.BHD can reduce Beclin-1 expression and LC3B-Ⅰ to LC3B-Ⅱ conversion in penumbra area of rats,and can also reduce the amount of autophagosome.BHDS can reduce Beclin-1 expression and LC3B-Ⅰ to LC3B-Ⅱ conversion in OGD/R group,reduce autophagosome fluorescence staining intensity and LC3 B fluorescence intensity,improve the OGD/R the neuron survival rate,and improve neurons form.Therefore,BHD can inhibit autophagy hyperactivation and reduce brain I/R injury.3.BHDS can reduce the levels of p-AMPK/AMPK,p-ULK1/ULK1,Beclin-1 and the fluorescence intensity of LC3 B in OGD/R groups,improve the survival rate of neurons and improve the morphology of neurons.Therefore,BHD can reduce brain I/R injury by regulating autophagy through AMPK/ULK1 signaling pathway. |
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