康复学报 (Apr 2022)
Effect of Berberine on the Inflammatory Response of ANA-1 Cells Induced by Angiotensin Ⅱ Based on TLR4 Signaling Pathway
Abstract
ObjectiveTo detect the effect and potential mechanism of berberine on the inflammatory response of ANA-1 cells induced by Angiotensin Ⅱ.MethodsANA-1 cells in logarithmic growth phase were randomly divided into the control group, the fluorescent antibody group, the model group, the low-dose BBR group, the medium-dose BBR group, the high-dose BBR group and the inhibitor group. The 2.5 μL dimethyl sulfoxide (DMSO) was added in the control group and the fluorescent antibody group; the concentration of 1 μmol/L Ang Ⅱ was added in the model group; the concentration of 1, 5, 10 μmol/L BBR was added in the low, medium and high-dose BBR groups; the concentration of 10 μmol/L TAK242 was added in the inhibitor group. Cell proliferation and toxicity test (CCK-8) was used to detect the effect of different concentrations of BBR on cell viability; the flow cytometry was used to detect the mean fluorescence intensity (MFI) of TLR4 on cell membrane ; ELISA method was used to detect the content of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α); the real-time quantitative PCR was used to detect the mRNA level of IL-1β, IL-6, TNF-α; Western blot was used to detect the phosphorylation of p-ERK/ERK, p-p38/p38, p-JNK/JNK, p-p65/p65 and p-IRF3/IRF3 and the expression of TLR4, MyD88 and AP-1; the biochemical assay was used to detect the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD).Results① CCK-8 assay results: compared with the control group, there was no significant difference in cell activity of the low, medium and high- dose BBR group, the difference was not statistically significant (P>0.05). ② Flow cytometry results: compared with the fluorescent antibody group, MFI of the model group was significantly decreased; compared with the model group, MFI of the model group + the high dose BBR group was significantly increased, the difference was statistically significant (P<0.05). ③ ELISA results: compared with the control group, the content of IL-1β, IL-6, TNF- α of the model group was significantly increased; compared with the model group, the content of IL-1β, IL-6, TNF- α of the model group + the low, medium and high dose BBR group was significantly decreased, the difference was statistically significant (P<0.05). ④ Real-time quantitative PCR results: compared with the control group, the mRNA level of IL-1β, IL-6, TNF-α of the model group was significantly increased; compared with the model group, the mRNA level of IL-1β, IL-6, TNF- α of the model group + the low, medium and high-dose BBR group was significantly decreased, the difference was statistically significant (P<0.05). ⑤ Western blot results: compared with the control group, the phosphorylation expression of p65 and IRF-3 protein of the model group after intervention for 45 minutes was significantly increased, and the phosphorylation expression of p65, IRF-3 protein in the model group + the low, medium and high-dose BBR group was significantly decreased. Compared with the control group, the phosphorylation expression of ERK, p38 protein of the model group after intervention for 15 minutes was significantly increased, the phosphorylation expression of JNK protein of the model group after intervention for 30 minutes was significantly increased, the phosphorylation expression of p65, IRF-3 protein of the model group after intervention for 45 minutes was significantly increased, the phosphorylation of p65, IRF-3, ERK, p38 and JNK proteins of the model + the high dose BBR group and the model + the inhibition group were significantly decreased. Compared with the control group, the expression of TLR4, MyD88, AP-1 protein and p65, IκBα, JNK protein phosphorylation of the model group after intervention for 12 h were significantly increased, and the expression of TLR4, MyD88, AP-1 protein and p65, IκBα, JNK protein phosphorylation of the model group + the low, medium, high-dose BBR group were significantly decreased. Compared with the control group, the expression of TLR4, p-p65 protein of the model group after intervention for 12 h was significantly increased, the expression of TLR4, p-p65 protein of the model + the high dose BBR group, the model group + the inhibitor group, the model group + the inhibitor group + the high dose BBR goup was significantly decreased. ⑥ Biochemical test results: compared with the control group, the content of MDA of the model group was significantly increased, SOD activity was significantly decreased, the difference was statistically significant (P<0.05), the content of MDA in the model group + the low, medium and high dose BBR group was significantly down-regulated, SOD activity was significantly increased, the difference was statistically significant (P<0.05).ConclusionBBR can regulate TLR4 signaling pathway and inhibit the abnormal activation of TLR4 signaling pathway induced by Ang Ⅱ and NF-κB, MAPK signal transduction, and reduce the content of proinflammatory factors and regulate inflammatory response; in addition, BBR can also improve the lipid peroxidation and antioxidant ability of ANA-1 cells induced by Ang Ⅱ, reduce the oxidative stress damage caused by Ang Ⅱ, and protect ANA-1 cells.
