ABSTRACT
Objective Bioinformatics analysis combined with cell experiment to explore the effect of Xintongtai medicated serum on apoptosis of rabbit aortic vascular smooth muscle cells by down-regulating PI3K/Akt/HIF-1α.Methods ox-LDL was used to induce the apoptosis of VSMC so as to establish the atherosclerotic cell model.The CCK8 method was used to select the optimal concentration of Xintongtai medicated serum.The main chemical components of Xintongtai were collected by TCMSP,the information of active compounds was collected by PubChem,the targets of active compounds were predicted by SwissTargetPrediction,the targets of"atherosclerosis"and"apoptosis"were collected by genecards and disgenet,and the protein-protein interaction(PPI)network was constructed by string platform.David online analysis gene ontology(GO)enrichment analysis function and Kyoto Encyclopedia of genes and genomes(KEGG)enrichment analysis.VSMC was divided into blank serum group,model group,Xintongtai medicated serum group and phosphatidylinositol 3 kinase(PI3K)inhibitor(LY294002)group,and Xintongtai medicated serum+ LY294002 group.The apoptosis of VSMC was detected by TUNEL and flow cytometry,and the apoptosis rate was calculated.The mRNA expression of PI3K,Akt,HIF-1α,caspase-3,caspase-9 were determined by polymerase chain reaction(PCR).Protein expression of p-PI3K/PI3K,p-Akt/Akt,HIF-1α,cleaved caspase-3,cleaved caspase-9 were determined by Western blot.α-SMA(Contractive VSMC specific marker)Fluorescence quantification of VSMC was determined by cellular immunofluorescence.Results The optimal concentration selected by CCK-8 was 20%middle dose Xintongtai medicated serum.Compared with the blank group,the VSMC early apoptosis rate,late apoptosis rate and total apoptosis rate in model group were increased(P<0.01),the mRNA expression of PI3K,Akt,HIF-1α,caspase-3,caspase-9 of the model group were up-regulated(P<0.01),the protein expression of p-PI3K/PI3K,p-Akt/Akt,HIF-1α,cleaved caspase-3,cleaved caspase-9 of the model group were up-regulated(P<0.01).Compared with the model group,the VSMC early apoptosis rate,late apoptosis rate and total apoptosis rate in Xintongtai medicated serum group,LY294002 group,and Xintongtai medicated serum+LY294002 group decreased(P<0.01 or P<0.05),and the mRNA expression of PI3K,Akt,HIF-1α,caspase-3,caspase-9 of Xintongtai medicated serum group,LY294002 group,and Xintongtai medicated serum+LY294002 group were down regulated(P<0.01 or P<0.05),the protein expression of p-PI3K/PI3K,p-Akt/Akt,HIF-1α,cleaved caspase-3,cleaved caspase-9 of Xintongtai medicated serum group,LY294002 group,and Xintongtai medicated serum+LY294002 group were down regulated(P<0.01 or P<0.05),the α-SMA increased significantly(P<0.01 or P<0.05).Compared with the Xintongtai medicated serum group,the VSMC early apoptosis rate,late apoptosis rate and total apoptosis rate were no difference(P>0.05),the mRNA expression of PI3K,Akt,HIF-1α,caspase-3,caspase-9 were no difference(P>0.05),the protein expression of p-PI3K/PI3K,p-Akt/Akt,HIF-1α,cleaved caspase-3,cleaved caspase-9 were no difference(P>0.05),the α-SMA was no difference(P>0.05).Conclusion Xintongtai medicated serum may down regulate PI3K/Akt/HIF-1α Signal pathway and downstream apoptosis related factors to alleviate the ox-LDL induced VSMC apoptosis,so as to stabilize vulnerable arterial plaque.
ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); host cell entry by this virus relies on the interaction between the receptor-binding domain (RBD) of its spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor on cell membranes. In addition to serving as a receptor for SARS-CoV-2, ACE2 was originally discovered as a protective factor in the renin-angiotensin system (RAS) that catalyses the degradation of angiotensin II (Ang II) to Ang 1-7, which is involved in multiple organ pathology. Recent genetic and clinical studies reported that ApoE4 expression is associated with increased susceptibility to SARS-CoV-2 infection and the development of severe COVID-19, but the underlying mechanism is currently unclear. In the present study, by using immunofluorescence staining, molecular dynamics simulations, proximity ligation assay (PLA) and coimmunoprecipitation (Co-IP) combined with a biolayer interferometry (BLI) assay, we found that ApoE interacts with both the spike protein and ACE2 but does not show obvious isoform-dependent binding effects. These data suggest that ApoE4 increases SARS-CoV-2 infectivity in a manner that may not depend on differential interactions with the spike protein or ACE2. Importantly, further immunoblotting and immunofluorescence staining results showed that ApoE4 significantly downregulates ACE2 protein expression in vitro and in vivo and subsequently decreases the conversion of Ang II to Ang 1-7, which could worsen tissue lesions; these findings provide a possible explain by which ApoE4 exacerbates COVID-19 disease.
ABSTRACT
Treatment of 34 cases of Simple ankle sprain mainly by Ashi point and in cooperation with magnetic therapy, and Qiuxu (GB 40), Shenmai (BL 62),Jinmen (BL 63) and Pucan ( BL 61 ) were added in the presence of strephenopodia sprain, Zhaohai (KI 6),Shuiquan (KI 5 ) and Taixi (KI 3 ) were added in the presence of strephexopodia sprain, the effective rate was 91.7%.
