Cell lysate samples were prepared using equivalent total protein concentrations, and analysed by employing western blotting. The blots were probed using primary antibodies generated against the following proteins p38, necessary signal regulated kinase, c Jun N terminal kinase, phosphorylated p38, phospho ERK, phospho JNK, NF ��B, and phospho p65. Primary antibody reactivity was visua lised using a horseradish pero idase conjugated secondary antibody and an enhanced chemiluminescence system. Statistical analyses Each e periment was replicated 6 times, and the data were presented as the mean standard deviation. Differ ences between the e perimental and control groups were analysed using the Mann Whitney U test, and P. 05 was considered to indicate a statistically significant inter group difference.
Results Sirolimus did not reduce the viability of the THP 1 cells The 24 h sirolimus treatment did not significantly change the viability of the THP 1 cells and primary monocytes, compared with the control group. Sirolimus suppressed lipopolysaccharide induced chemokine e pression in THP 1 cells and human primary monocytes Sirolimus significantly reduced the LPS induced e pression of MCP 1, RANTES, and IL 8 in the THP 1 cells and human primary mono cytes. In addition, Sirolimus significantly reduced the LPS induced e pression of MIP 1 in the THP 1 cells, whereas the e pression of both MIP 1 and MIP 1B was reduced in LPS treated human primary monocytes. The data sug gested that mTOR inhibition suppressed the e pression of nephrotic syndrome related chemokines in the THP 1 cells and human primary monocytes.
Sirolimus did not significantly reduce the LPS induced e pres sion of TNF i in THP 1 cells and human primary monocytes. Sirolimus suppressed lipopolysaccharide induced monocyte chemoattractant protein 1 e pression through mitogen activated protein kinase and nuclear factor ��B pathways in THP 1 cells Figure 5a and e indicate that SB203580, SP600125, and PD98059 suppressed the LPS induced e pression of MCP 1 and IL 8, suggesting that MAPK sig nalling is involved in the LPS induced e pression of MCP 1 and IL 8 in THP 1 cells. Figure 5b, d, and f show that the NF ��B inhibitor, BAY 11 7085, significantly re duced the LPS induced e pression of MCP 1, RANTES, and IL 8 in THP 1 cells, signifying that NF ��B inhibitor signalling is involved in the LPS induced e pression of MCP 1, RANTES, and IL 8 in THP 1 cells.
As shown in Figure 6a and c, SP600125 and PD98059 reduced the LPS induced e pression of MIP 1 and MIP 1B in THP 1 cells. SB203580 suppressed the LPS induced e pression of MIP 1B, but did not reduce the e pression Brefeldin_A of MIP 1 in THP 1 cells. Figure 6b and d show that BAY 11 7085 reduced the LPS induced e pression of MIP 1 and MIP 1B in THP 1 cells. Thus, and differentiate into macrophages and dendritic cells.