Effect Of TGF-β1on The Apoptosis Of Macrophage And The Relative Molecular Mechanisms

Object and contents:Transforming growth factor β1(TGF-β1) which is a member of the TGF superfamily plays an important role in the negative regulation of toll-like receptor (TLR) signaling in a feedback manner. It can induce the apoptosis of several types of cells, including but not limited to human B cells, B cell lymphoma, eosinophils, endothelial cells and hepatoma cells. The existing studies indicate that LPS stimulation contributes to the TGF-β1secreting by macrophages. Furthermore, LPS stimulation can promote macrophage apoptosis and the pro-apoptotic effect of LPS depends on activation of TLR4. However, It is still unclear whether LPS-induced endogenous TGF-β1is involved in the LPS-induced macrophage apoptosis. This work was performed to study the relationship between LPS-induced macrophage apoptosis and the generation of TGF-β by LPS-treated macrophages, to explore the effect of TGF-β1on the expression of TLR4on the surface of macrophages, and the related molecular mechanisms.Methods and results:The project is divided into three parts: Part One:Elucidate the role of TGF-β1in LPS-induced macrophage apoptosis.1. We detected the apoptosis of RAW264.7macrophage cells and the generation of TGF-β1by these macrophages after100and1000ng/ml LPS treatment for12hours. Results show that LPS treatment induced RAW264.7apoptosis and promoted the generation of TGF-β1in a dose dependent manner.2. We observed apoptosis of macrophages after5and lOng/ml TGF-β1treatment for12hours. Results show that TGF-β1treatment induced RAW264.7apoptosis in a dose dependent manner.3. We also detect the effect of TGF-β1on apoptosis of macrophages induced by LPS. We treated macrophages by100ng/ml LPS plus5or lOng/ml TGF-β1for12hours. We found that TGF-β1promoted LPS induced RAW264.7apoptosis in a dose dependent manner.Part Two:Detect the effect of TGF-β1on expression of TLR4on the surface of RAW264.7.1. LPS induced gene and protein expression of macrophage TLR4. We detected gene and protein expression of TLR4after macrophages were treated with100and1000ng/ml LPS for12hours. Results indicate that LPS inhibited the gene and protein expression of macrophage TLR4in a dose dependent way. We then stimulated RAW264.7with100ng/ml LPS and detected the gene and protein expression of TLR4at various time points. We observed a significant TLR4mRNA reduction at6hours. Between6to24hours, TLR4levels gradually recover to the basal levels.2. TGF-β1induced gene and protein expression of macrophage TLR4. We detected gene and protein expression of TLR4after macrophages were treated with5and lOng/ml TGF-β1treated macrophages for12hours. Results show that TGF-β1inhibited the gene and protein expression of macrophage TLR4in a dose dependent way. Second, we stimulated RAW264.7at different time points with lOng/ml TGF-β1and examined the gene and protein expression of TLR4. Results suggest that TGF-β1inhibited the gene and protein expression of macrophage TLR4in a time dependent manner.3. TGF-β1and/or LPS induced gene and protein expression of macrophage TLR4. The groups were as follows:Control,100ng/ml LPS, lOng/ml TGF-β1, LPS and TGF-β1mixture,1μg/ml anti-TGF-β1, LPS and anti-TGF-β1mixture. Results show that the TLR4gene and protein expression of the group treated with LPS and TGF-β1mixture was reduced at a greater rate compared to the group treated by LPS or TGF-β1. Anti-TGF-β1neutralizes TGF-β1secreted by macrophage, this reagent can partially abrogate but not completely block LPS to inhibit the expression of macrophage TLR4, it suggest that the inhibition of LPS on the expression of macrophage TLR4is not in TGF-β1-dependent manner.Part Three:Detect the effect of TGF-β1on related molecules in TLR4signal transduction. 1. Study the effect of TGF-β1or/and LPS on gene expression of macrophage SOCS3. Results show that LPS or TGF-β1treatment can promote SOCS3gene expression. SOCS3gene expression increased in the LPS+anti-TGF-β1group but was not markedly different from the LPS group. There is no significant difference between the LPS group and LPS+anti-TGF-β1group on the expression of SOCS3.2. Detection of the effect of TGF-β1or/and LPS on the gene expression of macrophage BAMBI. Results indicate that LPS or TGF-β1treatment can inhibit BAMBI gene expression. There is no significant difference between the LPS group and LPS+TGF-β1group on the expression of BAMBI. BAMBI gene expression decreased in the LPS+anti-TGF-β1group but was not markedly different from the LPS group.Conclusion:1. LPS-induced macrophage apoptosis correlates with the generation of TGF-β by LPS-treated macrophages. TGF-β1treatment can induce RAW264.7apoptosis. Combined LPS and TGF-β1treatment enhances the apoptosis of RAW264.7.2. LPS inhibition of gene and protein expression of macrophage TLR4is correlated to the generation of TGF-β1by LPS-treated macrophages, TGF-β1can inhibit the the gene and protein expression of macrophage TLR4. Anti-TGF-β1blocking antibody can partially overcome but not completely block LPS to inhibit the expression of macrophage TLR4, it suggest that the inhibition of LPS on the expression of macrophage TLR4is not in TGF-β1-dependent manner.3. TGF-β1or/and LPS treatment can promote the gene expression of macrophage SOCS3and inhibit the gene expression of macrophage BAMBI. We have not yet observed a synergistic effect between LPS and TGF-β1on regulating the gene expression of SOCS3and BAMBI in this experiment.