Mechanism Of Longzhi Decoction-containing Serum Promoting Human Umbilical Vein Endothelial Cell Angiogenesis In Vitro Via PI3K/Akt/mTOR Pathway

Objective:We observed the effect of Longzhi Decoction-containing serum（LZD）on the autophagy and apoptosis of HUVECs induced by hydrogen peroxide（H₂O₂）oxidative stress,and explored the possible molecular mechanism of LZD promoting the formation of HUVECs.Methods:According to the method of serum pharmacology,Longzhi decoction-containing serum and Butylphthalide-containing serum were prepared,and H₂O₂-induced oxidative stress autophagy model of HUVECs was constructed in vitro.In the first experiment,the interaction between the target of Chinese medicine components of Longzhi Decoction predicted by network pharmacology and the target of ischemic stroke disease was constructed,and the obtained target was analyzed for GO function enrichment and KEGG enrichment pathway.The experiment was divided into 1.Normal control group（Control）;2.Blank serum group（BSG）;3.H₂O₂ group（H₂O₂）;4.Butylphthalide-containing serum group（NBP）;5.LZD group;6.3-MA+H₂O₂;7.3-MA+H₂O₂+LZD group;8.Rapa+H₂O₂ group;9.Rapa+H₂O₂+LZD group.The cell proliferation,cell migration,and tube formation ability of HUVECs were detected by CCK-8,scratch test,and matrigel tube formation test respectively;and the fluorescence intensity of autophagy was detected by cellular immunofluorescence.RT-q PCR was used to detect the expression of LC3 II/I,Beclin-1,VEGF,and mTOR m RNA in each group.Western blot was used to quantify LC3 II/I,Beclin-1,Bcl-2,Bax,VEGF protein,and phosphorylated PI3K and Akt proteins.At the same time,by applying RNA interference technology,Beclin-1 was constructed from lentiviral vectors to silence HUVECs,and the molecular regulation mechanism of PI3K/Akt/mTOR signaling pathway involved in HUVECs angiogenesis was studied.Results:（1）Compared with the Control group,the cell viability of the H₂O₂group decreased significantly,and the cell viability of the LZD and NBP groups increased significantly.Compared with H₂O₂,the cell migration number and migration area of the LZD and NBP groups increased significantly;meanwhile,the cell connectivity within the tubular structure was more complete and the branch points increased and the number of tube formations increased significantly.（2）The number of surviving HUVECs cells decreased with time and concentration,which was time and concentration dependent.（3）The results of network pharmacological analysis showed that Longzhi Decoction has 145compounds,and 176 targets were obtained after screening.The compound component-ischemic stroke disease-target network predicts 17 common targets.The cytological components are significantly expressed by receptor complexes,plasma membrane protein complexes,and neuronal cell membranes.The molecular biological functions are mainly peptidase activity,oxidoreductase activity,and hormone binding.The biological process involves apoptosis signaling pathway,positive regulation of kinase activity,regulation of blood coagulation,etc.The KEGG enrichment signaling pathway involves PI3K-Akt signaling pathway,HIF-1 signaling pathway,cytokine-mediated signaling pathway,response to oxygen levels,positive regulation of cell migration and blood circulation.（4）Compared with the 3-MA and Rapa group,the cell proliferation rate of the two groups of drugs was increased after adding LZD;and with the increase of the concentration of 3-MA and Rapa,the increase of the cell proliferation rate was smaller（P<0.05）.（5）The fluorescence intensity of the Control group and the 3-MA group is the weakest,and the fluorescence intensity of the Rapa group is the strongest.Compared with the 3-MA group,the fluorescence expression level of the 3-MA+LZD group increased significantly;compared with the Rapa group,the fluorescence expression level of the Rapa+LZD group decreased（P<0.05）.（6）Compared with the H₂O₂ group,the expression levels of LC3 II/I and Beclin-1 in the 3-MA group were reduced,and the expression levels of Bcl-2 and Bax were increased;the 3-MA+LZD group could reverse the effects caused by 3-MA The expression level of LC3I II/I and Beclin-1 protein decreased,while the expression level of Bcl-2 protein was increased and the expression level of Bax protein was decreased（P<0.05）.The expression levels of LC3 II/I,Beclin-1,Bax protein in Rapa group were significantly increased,and the expression level of Bcl-2 protein was significantly decreased;while the levels of autophagy and apoptosis protein in Rapa+LZD group were decreased,and the level of Bcl-2 protein was increased（P<0.05）.（7）Compared with 3-MA group and Rapa group,the levels of p-PI3K,p-Akt and VEGF protein in 3-MA+LZD group and Rapa+LZD group increased,and the relative expression of mTOR and VEGF m RNA increased significantly（P<0.05）.（8）Compared with the H₂O₂ group,the cell migration number and migration area of the 3-MA group and Rapa group were reduced;the cell migration number and migration area of the 3-MA+LZD group and Rapa+LZD group were respectively Compared with the 3-MA group and the Rapa group,it increased significantly（P<0.05）.（9）Compared with the H₂O₂group,the number of casts in the 3-MA group and the Rapa group decreased.After co-treatment of HUVECs with LZD,the phenomenon of reduced casts was reversed.（10）The negative control of the lentiviral transfection Con-Beclin-1+H₂O₂ group had strong red-green fluorescence expression,while the red fluorescence of the Con-Beclin-1+H₂O₂+LZD group weakened,mainly green fluorescence expression;the positive control of lentivirus transfection In the Sh-Beclin-1 group,the expression of green fluorescence was dominant,and the expression of red fluorescence was minimal.After adding LZD,the red fluorescence did not increase or decrease significantly（P<0.05）.（11）The expression levels of p-PI3K,p-Akt and VEGF protein in Con-Beclin-1+H₂O₂group were low.After adding LZD to intervene in HUVECs,the expression levels of the above factors could be significantly increased（P<0.01）.In addition,Beclin-1 m RNA expression decreased,mTOR,VEGF m RNA expression increased significantly.Compared with Con-Beclin-1+H₂O₂+LZD group,the expression levels of p-PI3K,p-Akt and VEGF protein in Sh-Beclin-1+H₂O₂group and Sh-Beclin-1+H₂O₂+LZD group were significantly reduced（P<0.01）,and Beclin-1,mTOR,VEGF m RNA expression also decreased simultaneously.Compared with the expression levels of the above factors in the Sh-Beclin-1+H₂O₂ group and the Sh-Beclin-1+H₂O₂+LZD group,there was no statistically significant difference between the groups（P>0.05）.Conclusion:（1）Based on network pharmacology,construct Longzhi Tang compound-target network of ischemic stroke disease,Longzhi Decoction may participate in transduction of PI3K by acting on Akt-1,Bcl-2,ANGPT1,PTGS2and other targets PI3K/Akt signaling pathway,HIF-1 signaling pathway,response to oxygen levels,apoptosis signaling pathway,etc.And regulate the biological processes of cell proliferation,migration and apoptosis.（2）LZD can effectively improve cell vitality and help promote cell migration and cast formation.（3）LZD may regulate the autophagy factor by activating the PI3K/Akt/mTOR signaling pathway,reduce cell apoptosis,up-regulate the level of VEGF,and stimulate the activation mechanism of angiogenesis factors,which play a role in promoting angiogenesis in vitro,which may have potential bidirectional regulation.