Berberine Regulates Angiotensin Ⅱ-induced Inflammatory Response In ANA-1 Cells Through TLR4 Signaling Pathway

ObjectiveTo study the effect of berberine on the inflammatory response induced by Angiotensin Ⅱ in ANA-1 cells and its potential mechanism.Method1.ANA-1 macrophages were cultured,CCK-8 method was used to detect the effect of different concentrations(0,0.1,0.5,1,5,10,50 μm)of BBR on the viability of ANA-1 cells,and this was used as the concentration selection for subsequent experiments.2.The mean fluorescence intensity of TLR4 on cell membrane was detected by flow cytometry.The experiment was divided into four groups: blank group,TLR4/PE group,Ang Ⅱ+ TLR4/PE group,BBR + Ang Ⅱ + TLR4/PE group.3.Q-PCR was used to detect the m RNA expressions of IL-1β,IL-6 and TNF-α.4.ELISA method was used to detect the contents of pro-inflammatory cytokines IL-1 β,IL-6 and TNF-α.5.Western blot was used to detect the phosphorylation of p-ERK/ERK,p-p38/p38,p-JNK/JNK,p-p65/p65,p-IRF3/IRF3 and the expression of TLR4,My D88 and AP-1.6.Biochemical test was used to detect the content of MDA and the activity of SOD.Result1.CCK-8 assay results showed that: compared with the control group,the cell viability of 50 μ M group decreased significantly,the difference was statistically significant(P < 0.05),while the other groups had no significant change,the difference was not statistically significant,indicating that BBR concentration in the range of 0.5-10 μm had no obvious toxicity to ANA-1 cells,which can be used as the concentration of the next experiment.2.The results of flow cytometry showed that compared with TLR4/PE group,the average fluorescence intensity of TLR4 in Ang Ⅱ group was significantly decreased,while the average fluorescence intensity of BBR + Ang Ⅱ group was significantly increased,indicating that BBR can up regulate the expression of TLR4 on cell membrane by inhibiting the endocytosis of TLR4.3.The results of Q-PCR showed that the m RNA levels of IL-1β,IL-6 and TNF-α in Ang Ⅱ group were significantly up regulated compared with the control group.After BBR and TAK242,the m RNA levels of IL-1β,IL-6 and TNF-α induced by Ang Ⅱ were significantly reduced,and the influence of BBR on these inflammatory factors was concentration dependent.4.The results of ELISA detection showed that compared with the control group,the contents of IL-1β,IL-6,TNF-α and other pro-inflammatory factors in the supernatant of Ang Ⅱ group were significantly increased,while BBR and TAK242 could reduce the content of these pro-inflammatory factors,and the effect of BBR on these pro-inflammatory factors was in a dose-dependent manner.5.Western blot results showed that: compared with the control group,Ang Ⅱ could significantly up regulate the phosphorylation expression of p-ERK/ERK and p-p38/p38 after stimulation for 15 min;significantly up regulate the phosphorylation expression of p-JNK/JNK after stimulation for 30 min;significantly up regulate the phosphorylation expression of p-p65/p65 and p-IRF3/IRF3 after stimulation for 45 min;significantly up regulate the expression of TLR4,My D88,AP-1 and phosphorylation of p-IκBα/IκBα 、p-p65/p65 、 p-JNK/JNK after stimulation for 12 h.BBR or TAK242 pretreatment for 2 h significantly down regulated the expression of p-ERK/ERK,p-p38/p38,p-JNK/JNK,p-p65/p65,p-IRF3/IRF3,P-IκBα/IκBα phosphorylation and TLR4,My D88,AP-1 protein expression induced by Ang Ⅱ in a dose-dependent manner.6.The results of biochemical test showed that: compared with the control group,the MDA content in Ang Ⅱ group was significantly increased,indicating that Ang Ⅱ can increase the degree of lipid peroxidation of ANA-1,while BBR can reduce the MDA content and improve the oxidative stress injury of ANA-1 induced by Ang Ⅱ.Moreover,compared with the control group,the SOD activity of Ang Ⅱ group was significantly decreased,indicating that the stimulation of Ang Ⅱ reduced the antioxidant capacity of ANA-1 cells,while BBR could significantly improve the antioxidant capacity of ANA-1 cells and reduce oxidative stress damage.ConclusionBBR can inhibit the abnormal activation of TLR4 signaling pathway induced by Ang Ⅱ by inhibiting the endocytosis of TLR4.Meanwhile,BBR can inhibit the activation of NF-κB and MAPK signaling pathway downstream of TLR4 signaling pathway,and reduce the levels and contents of IL-1β,IL-6 and TNF-α.In addition,BBR can improve the degree of lipid peroxidation and antioxidant capacity of ANA-1 cells induced by Ang Ⅱ,reduce the oxidative stress injury caused by Ang Ⅱ,and protect ANA-1 cells.