Research Of Adipocyte Stimulated By Ffa And Activate Toll-nf-kb Signal Pathway Mechanism

Objective:Excess adipose tissue accumulation in obesity is tendency to lipolysis. It makes the increasing of plasma FFA and the accumulation of fat in cell. Obesity is considered as a kind of slow inflammation state. First,3T3-l1 preadipocyte are cultivated and then induced into mature adipose cell. FFA of fixed concentration is used to stimulate adipose cell. Then it is tested that whether FFA can influence the expression of TLR4 and induce NF-κB signal passage furtherly and at last strengthen the secretion of inflammation adipocyte.Methods:3T3-L1preadipocyte is induced mature adipose cell(Red oil O to identify). LPS, EPA, FFA act on mature adipose cell (LPS as the positive control, EPA as the negative control). The totol protein of The cells are collected and their TLR4 and NF-κB protein express are tested using Western Blot. the cells are collected and their TLR4 mRNA express are tested using Real-time PCR. nucleus protein is extracted and the actieness of NF-κB in each group are tested using EMSA. culture medium collected and the expression of cytokine of TNF-α, IL-6, MCP-1 are tested using EMSA. The date was analysed with statistics software of the SPSS 13.0.According to these results, it can be judged that whether FFA can influence the expression of TLR4 and induce NF-κB inflammation signal passage through TLR4.Results:1. In this research, the 3T3-L1preadipocyte is induced into adipose cell and put into study. Generally acknowledged,3T3-L1 is a model of cell which is used to study the split of cells. Our researchers were skillful at differentiation method.ensure the differentiation ratio of mature adipocyt were stable.2. Through the expression of mRNA in TLR4 after being tested using Real-time PCR, it can be concluded that the relative mRNA of FFA is 2.1, EPA is 1.2, LPS is 2.7. The relative mRNA of blank control is 1. Certain stimulation of FFA can make the expression of mRNA lower than the positive control, higher than the negative control and blank control.3. FFA of certain concentration is used to stimulate adipose cell, and then positive control with LPS, negative control with EPA and blank control. After testing using Western-blot, it can be seen that the expression of protein of TLR4 is lower than the positive control and higher than the negative control and blank control.4. FFA of certain concentration is used to stimulate adipose cell, and then positive control with LPS, negative control with EPA and blank control.After testing using Western-blot, it can be seen that the expression of NF-κB on the level of protein of TLR4 is lower than the positive control and higher than the negative control and blank control.5. FFA of certain concentration is used to stimulate adipose cell, and then positive control with LPS, negative control with EPA and blank control. After testing the transcribe activity of NF-κB using EMSA, it can be concluded that stimulation of FFA of certain concentration can increase the transcribe activity of the adipose cell NF-κB.6. FFA of certain concentration is used to stimulate adipose cell, and then positive control with LPS, negative control with EPA and blank control. Concentration and tendency of change of MCP-1 in culture medium are tested using ELISA. The result shows that the concentration of MCP-1 in group FFA and LPS are the same at first and with obvious rising after 2hours. The concentration of MCP-1 in group EPA and blank control are the same. Difference of the concentration of MCP-1 are exist at 4h,6h,8h,10h,12h (P<0.05) between group FFA and blank control.7. FFA of certain concentration is used to stimulate adipose cell, and then positive control with LPS, negative control with EPA and blank control. Concentration and tendency of change of IL-6 in culture medium are tested using ELISA. Tendency of concentration of IL-6 is LPS>FFA> blank control>EPA. There is no difference of concentration of IL-6 in group FFA and blank control before 8h (P>0.05) and the difference appears at the 10h hour (P<0.05). At the 10h hour, IL-6 in group LPS group is 224.03±18.96pg/mL, FFA group is 199.13±3.55 pg/mL. blank control is 171.83±6.47 pg/mL, EPA group is 126.20±4.35 pg/mL, FFA group compared with blank control P＜0.05.8. FFA of certain concentration is used to stimulate adipose cell, and then positive control with LPS, negative control with EPA and blank control. Concentration and tendency of change of TNF-a in culture medium are tested using ELISA. The concentration of TNF-a in group LPS is obviously higher than the other three groups but in group EPA and blank control, it is the same. Difference of the concentration of TNF-a are exist at 4h,6h,8h,10h,12h(P<0.05) between group FFA and blank control.Conclusion:1.Stimulation of FFA of certain concentration on adipose cell can strengthen the expression of mRNA level and protein level of TLR4 receptor.2.Adipocyte Stimulated by FFA induct the NF-κB inflammention signal pathway.3.Secreting concentration of IL-6,TNF-a,MCP-1, which is the gene expression product of NF-κB, also can be increased to varying degrees in adipocyte after stimulated by FFA.