Effect Of Huoxue Dingxuan Capsule On CSA Model Rats Through PI3K/Akt/mTOR Signaling Pathway

ObjectiveIn this study,we cultured bEnd in vitro.3 cells and physical compression combined with mechanical imbalance simulate the ischemic and hypoxic state of cervical spondylosis of vertebral artery type(CSA),and observe the changes of autophagy in vascular endothelial cells under hypoxic ischemia and the intervention effect of Huo-Xue-Ding-Xuan-Capsule(HXDXC),providing an experimental basis for the further clinical application of HXDXC.Methods1.in vitro culture bEnd.3 cells,randomly divided into 7 groups: normal control group,model group,10% HXDXC medicated serum group,LY294002 group,rapamycin group,LY294002 + 10% HXDXC medicated serum group,rapamycin + 10% HXDXC medicated serum group,oxygen glucose deprivation(OGD)model was established by hypoxic culture in addition to normal control group,PI3K/Akt/mTOR signaling pathway inhibitor LY294002 and rapamycin and 10% concentration HXDXC medicated serum were applied for intervention,and then bEnd of each group was observed by transmission electron microscopy.3 cell autophage;Ad-mCherry-GFP-LC3 B recombinant adenovirus transfection confocal laser scanning was used to observe LC3 B expression;Western blot technique was used to detect bEnd in each group.3 Protein expression such as LC3 B,Beclin1,and p62 in cells and the expression of p-PI3K/PI3 K,p-Akt/Akt,and p-mTOR/mTOR after addition of inhibitors LY294002 and rapamycin.qRT-PCR technique was used to detect bEnd in each group.Relative Beclin1,ATG5,PI3 K,Akt,and mTORmRNA expression in 3 cells.The above experiments were performed to observe the effect of 10% concentration of HXDXC medicated serum on bEnd.3 Effect of autophagy in cells.2.80 Wistar male rats were randomly divided into 8 groups,10 rats in each group.The specific groups were:normal control group,sham operation group,model group,HXDXC high(1.26 g/kg),medium(0.63 g/kg),low(0.315 g/kg)dose groups,flunarizine group(1.05mg/kg),and Jingfukang group(1.05 g/kg).Except nomol control group and sham operationgroup,other groups were used to establish CSA rat models by compression combined with mechanical imbalance method.After successful modeling,the animals were fed continuously for 6 weeks,after which the animals were anesthetized,the hearts were bled,sacrificed by cervical dislocation,and the vertebral arteries were removed.The body weight and water intake of rats were measured at different time points before sacrifice,and the response and spatial recognition ability of rats were observed by Morris water maze test;ELISA methods were used to detect the concentration of nitric oxide(NO),the content of vascular endothelin-1(ET-1)and the activity of superoxide dismutase(SOD)in the serum of rats in each group;transmission electron microscopy was used to observe vertebral artery vascular endothelial autophagosomes;immunohistochemistry was used to detect the expression of p65,Beclin1,and LC3 B protein in vertebral artery vascular endothelial tissues;immunofluorescence was used to count the punctate aggregation of LC3 in vertebral artery vascular endothelial cells tissues;Western blot was used to detect the expression levels of autophagy-related proteins Beclin1,LC3 B,and p62 protein;qRT-PCR was used to detect the expression of autophagy-related genes ATG5,Beclin1,and LC3 mRNA.Through the above in vivo experiments,the regulatory effects of different doses of HXDXC on autophagy in vertebral artery vascular endothelial cells of CSA model rats were observed,and the optimal dose of HXDXC for intervention of autophagy was determined.3.On the basis of experiment 2,the optimal dose of HXDXC group was selected,and the CSA rat model was established by compression combined with mechanical imbalance method.After successful modeling,the rats were randomly divided into 5 groups and intervened with PI3 K inhibitor LY294002 and mTOR inhibitor rapamycin and HXDXC in the optimal dose group,respectively.The specific groups were: model group,LY294002 group,rapamycin group,LY294002 + HXDXC optimal dose group,and rapamycin + HXDXC optimal dose group.After 6 weeks of continuous feeding,the animals were anesthetized,the hearts were bled,sacrificed by cervical dislocation,and the vertebral arteries were removed to observe the autophagic bodies of vertebral artery vascular endothelial cells using transmission electron microscopy,and the punctate accumulation of LC3 in vertebral artery vascular endothelialcells was counted by immunofluorescence;the expression levels of autophagy-related genes Beclin1,LC3 B,P62,and p-P13K/PI3 K,p-Akt/Akt,and p-mTOR/mTOR proteins were detected by Western blot;and the relative expression levels of autophagy-related genes ATG5,Beclin1,LC3 mRNA,and PI3 K,Akt,and mTORmRNA were detected by qRT-PCR.Results1.bEnd.3 After 6 hours of anaerobic culture,the cells were intervened by PI3 K inhibitor LY294002,mTOR inhibitor rapamycin and 10% HXDXC containing serum.The results showed that under transmission electron microscope,a large number of vacuolar bilayer membrane structures were observed in the model group and 10% HXDXC containing serum rapamycin group,surrounded by autophages,while LY294002 containing serum was less in the normal control group;after Ad-mCherry-GFP-LC3 B recombinant adenovirus transfection,a large number of yellow spots were found in the model group and 10% HXDXC containing serum rapamycin group under confocal laser scanning,while LY294002 group had fewer yellow spots,including rapamycin + 10% HXDXC containing serum rapamycin group;LC3II/LC3 I and Beclin1 expression was increased in the rapamycin group,i.e.The expression of p-mTOR/mTOR,p-Akt/Ak,and P62 was decreased(P < 0.05);the expression of LC3II/LC3 I,Beclin1,and p-PI3K/PI3 K was increased in the LY294002 + 10% HXDXC medicated serum LY294002 group compared with the model group(P < 0.05);the expression of LC3II/LC3 I and Beclin1 was decreased and the expression of P62,p-Akt/Akt,and p-mTOR/mTOR was increased in the rapamycin + 10% HXDXC medicated serum rapamycin group compared with the model group(P < 0.05);the qRT-PCR results showed that the expression of Beclin1,ATG5,and PI3 K mRNA was decreased in the LY294002 group compared with the model group(P < 0.05),and the expression of Beclin1 and ATG5 was increased and the expression of mTORmRNA was low in the rapamycin group(P < 0.05).2.In vivo animal experiment,the effects of different doses of HXDXC on CSA rats were observed.HXDXC could improve the physiological status and improve the memory and spatial recognition ability of CSA rats;after modeling,the NO concentration and SOD activity in the serum of rats were decreased,and the ET-1 content was increased;after drugintervention,the NO concentration and SOD activity were increased,and the ET-1 content was decreased in the HXDXC high-dose group,flunarizine group,and Jingfukang group,indicating that the blood vessels were damaged and contracted and the oxidative activity was decreased after modeling by compression combined with mechanical imbalance in rats,while HXDXC could significantly regulate vasoconstrictor factors and oxidative active substances.The results of transmission electron microscopy showed that a large number of autophages were observed in the vascular endothelial cells of the vertebral artery in the model group,while the autophages were significantly reduced in the HXDXC high-dose group;immunofluorescence confocal laser scanning assay revealed a large number of LC3 aggregation points in the model,while the LC3 aggregation points in the HXDXC high-dose group were reduced,and the fluorescence gray value analysis was statistically significant(P <0.05).Immunohistochemical results showed that compared with the nomol control group,the expression of p65,Beclin1 and LC3 B in the vertebral artery tissue of the model group was higher(P < 0.05);compared with the model group,the expression of p65,Beclin1 and LC3 B in the HXDXC high-dose group was decreased(P < 0.05),and there was no significant difference compared with the Jingfukang and flunarizine groups(P < 0.05);Western blot results showed that compared with the nomol control group,the expression levels of Beclin1 and LC3II/LC3 I in the model group were increased,and the expression levels of p62 were decreased(P < 0.05);compared with the model group,the expression levels of Beclin1 and LC3II/LC3 I in the HXDXC high-dose group,flunarizine group,and Jingfukang group were decreased,and the expression levels of p62 were increased(P < 0.05).qRT-PCR results showed that: compared with the nomol control group,the expression levels of ATG5,Beclin1,and LC3 mRNA in the model group were increased(P < 0.05);compared with the model group,the expression levels of ATG5,Beclin1,and LC3 mRNA in the HXDXC high-dose group,flunarizine group,and Jingfukang group were decreased(P < 0.05).3.The animal model of CSA was established by compression combined with mechanical imbalance,after high-dose intervention with PI3 K inhibitor LY294002,mTOR inhibitor rapamycin and HXDXC.The results showed that there were a large number of autophages inthe vascular endothelial cells of the vertebral artery in the model rapamycin group,while LY294002 was less.Immunofluorescence detected the number of punctate aggregates of LC3 and found that a large number of aggregation points were observed in the model rapamycin,but less in LY294002(P < 0.05);the number of punctate aggregates of LC3 was increased in the LY294002 + HXDXC high-dose LY294002 group,and decreased in the rapamycin +HXDXC high-dose rapamycin group,and the difference was statistically significant in the analysis of fluorescence gray values(P < 0.05).Western blot results showed that compared with the model group,the expression of LC3II/LC3 I,Beclin1,p-PI3K/PI3 K,and p-Akt/Akt was decreased and the expression of p62 was increased in the LY294002 group(P < 0.05),and the expression of LC3II/LC3 I and Beclin1 was increased and the expression of p-Akt/Akt and p-mTOR/mTOR was decreased in the rapamycin group(P < 0.05);the expression of LC3II/LC3 I,Beclin1,and p-PI3K/PI3 K was increased in the LY294002 + HXDXC high-dose LY29002 group,and the expression of LC3II/LC3 I and Beclin1 was decreased and the expression of p62 and p-mTOR/mTOR was increased in the rapamycin + HXDXC high-dose rapamycin group(P < 0.05).qRT-PCR results showed that: compared with the model group,the expression of Beclin1,LC3,ATG5,and PI3 K mRNA was decreased in the LY294002 group,and the expression of Beclin1,LC3,and ATG5 mRNA was increased and the expression of mTORmRNA was decreased in the rapamycin group;the expression of Beclin1,LC3,ATG5,and PI3 K mRNA was increased in the LY294002 + HXDXC high-dose LY29002group(P < 0.05);and the expression of Beclin1,LC3,and ATG5 mRNA was decreased and the expression of mTORmRNA was increased in the rapamycin + HXDXC high-dose rapamycin group(P < 0.05).Conclusions1.in vitro hypoxic culture of mouse brain microvascular endothelial bEnd.3 cell.After 6hours,autophagy could be activated.After intervention with the PI3 K inhibitor LY294002,the degree of autophagy was attenuated;after intervention with the mTOR inhibitor rapamycin,the degree of autophagy was enhanced.However,10% HXDXC medicated serum after intervention with LY294002 inhibitor.bEnd.3 cell autophagy had a promoting effect,after rapamycin intervention.Autophagy in bEnd3 cells has an inhibitory effect.The above results indicate that PI3K/Akt/mTOR signaling pathway plays a key role in regulating the occurrence and development of autophagy after tissue ischemia and hypoxia,while 10%HXDXC medicated serum has a significant effect on bEnd.3 cells Autophagy,It plays a bidirectional regulatory role.2.Autophagy occurred in rat vertebral artery vascular endothelial cells after the animal model of CSA was established by compression combined with mechanical imbalance.However,after drug intervention,autophagy was inhibited to different extents,and the activity of vascular endothelial cells was greatly improved compared with the model,in which the effect of high dose of HXDXC was better,indicating that after the blood vessels were compressed ischemia and hypoxia,the vascular endothelium was damaged and autophagy phenomenon occurred,while HXDXC may regulate vascular endothelial function by inhibiting autophagy in vertebral artery vascular endothelial cells of CSA rats.3.After the intervention of PI3 K inhibitor LY294002 and mTOR inhibitor rapamycin,autophagy of vertebral artery vascular endothelial cells was inhibited or activated to different extents in CSA model rats,while after the intervention of high dose of HXDXC,autophagy of vertebral artery vascular endothelial cells showed that HXDXC could increase the intensity of autophagy when autophagy was deficient,and played an appropriate role in inhibiting autophagy transition.HXDXC,a function that regulates autophagy,may be one of the mechanisms by which it protects vascular endothelial cell function.