Regulation of input protein α3/NF-κB pathway by adjusting miR-181 with dissipating phlegm and removing blood stasis method and its anti-atherosclerosis mechanism
JOURNAL OF BEIJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
2020, 43 (8):
Objective To observe the correlation between the anti-atherosclerosis mechanism of dissipating phlegm and removing blood stasis method and the effect on the input protein α3/NF-κB signaling pathway through the regulation of microribonucleic acid 181 (miR-181).
Methods In vitro experiments, vascular endothelial cell injury model was established with oxidized low-density lipoprotein (ox-LDL). According to different intervention methods, cells were randomly divided into four groups: model group (blank serum), miR-181 mimic group, miR -181 inhibitor group, phlegm-resolving and blood stasis-eliminating TCM serum group (TCM serum group). After intervention, the cell activity of each group was detected. RT-PCR and Western blot were used to detect the mRNA and protein expression levels of miR-181, input protein α3 and NF-κB. In vivo experiments, 60 ApoE
-/- mice were randomly divided into 5 groups: model group, miR-181 inhibitor group, miR-181 mimic group, high-dose and low-dose phlegm-resolving and blood stasis-eliminating TCM serum group (TCM high-dose serum group, and TCM low-dose serum group respectively) (
n=12). After continuous intervention for 8 weeks, the plasma and aorta were collected for testing. The levels of IL-6, VCAM-1 and E-selectin in the peripheral plasma were tested by ELISA. The expression of microRNA-181, input protein α3 and NF-κB in the aortic wall of each group was tested by RT-PCR, and the pathological changes were observed.
Results In vitro experiments, the cell activity of the TCM serum group and the miR-181 mimic group was significantly increased (
P<0.05), and the expression of α3 and NF-κB mRNA were significantly down-regulated (
P<0.05). In vivo experiments, compared with the model group, the plasma levels of IL-6, VCAM-1 and E-selectin in the TCM high-dose and low-dose serum groups and miR-181 mimic group were significantly lower, compared with the model group (
P<0.05); compared with the inhibitor group, the levels of peripheral plasma IL-6, VCAM-1 and E-selectin in the TCM serum group were lower (
P<0.05). Compared with the model group, the mRNA expression of aortic miR-181 in the TCM high-dose and low-dose serum groups and miR-181 mimic group was significantly increased (
P<0.05), and the expression level of α3 and NF-κB significantly down-regulated (
P<0.05). There was no significant difference in the indexes between the TCM high-dose and low-dose serum groups (
Conclusion The anti-atherosclerosis mechanism of resolving phlegm and eliminating blood stasis may be related to the targeted regulation of miR-181 to affect the input protein α3/NF-κB signaling pathway, which could possibly reduce the levels of inflammatory mediators such as IL-6, VCAM-1 and E-selectin, thus reducing the damage of vascular endothelial cells.
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