Fig 1.
The levels of C5a, IL-6 and TNF-α protein in the renal tissues of rats with Thy-1N.
(A and B) Rat Thy-1N was induced and then the protein level of C5a in the rat renal tissues was detected at different time points (0 h, 0.5 h, 1 h, 2 h and 3 h; n = 4 in each time point) after Thy-1N induction by Western blot assay. (C-E) The mRNA and protein levels of IL-6 and TNF-α in the renal tissues of Thy-1N rats was detected at various time points (0 h, 1 h, 2 h, 4 h, 8 h and 12 h; n = 6 in each time point) after Thy-1N induction by RT-PCR (C and D) and ELISA (E) respectively. Results were represented as means ± SD. Representative photographs were shown. ** P<0.01 versus 0 h time point (non-treated).
Fig 2.
Recombinant rat C5a identification and its biological activity analysis.
(A) Western blot was used to detect the recombinant rat His-C5a protein by the antibody to His-tag. (B) Neutrophils were stimulated with C5a (50 ng/ml) for 5 min, and then incubated with DCFH-DA (10 μM) for 30 min. Subsequently, flow cytometry was performed to detect the percentage of DCFH-DA-positive neutrophils. (C) Transwell assay was used to detect neutrophil chemotaxis induced by C5a stimulation at the dose of 50 ng/ml for 30 min. The neutrophils on the membrane were observed by crystal violet staining under the microscope. Representative photographs were manifested (n = 3 in each group).
Fig 3.
The expression of IL-6 and TNF-α in rat GMC stimulated with C5a.
(A and B) RT-PCR analysis was performed to measure the mRNA levels of IL-6 and TNF-α in rat GMC exposed to C5a (50 ng/ml) for different time points (0 h, 1 h, 2 h, 4 h, 8 h and 12 h). (C) ELISA assay was done to detect IL-6 and TNF-α release from the GMC upon C5a stimulation (50 ng/ml) for above-mentioned time points. (D) ELISA experiments were performed to detect IL-6 and TNF-α release from the GMC stimulated with C5a at the dose of 50 ng/ml or with endotoxin at the dose of 0.001 EU/ml or 50 EU/ml for 8 h. ** P<0.01 versus 0 h time point (non-treated), ns P>0.05 versus MEM group, # P<0.05 versus MEM group. Results were represented as means ± SD (n = 3 in each time point). Representative photographs were displayed.
Fig 4.
C5aR expression on rat GMC stimulated with C5a.
(A and B) Western blot assay was performed to determine the expression of C5aR on the GMC incubated with C5a (50 ng/ml) for different time points (0 h, 4 h, 8 h and 12 h). Results were represented as means ± SD (n = 3 in each time point), and representative photographs were exhibited.
Fig 5.
The phosphorylation of p38 MAPK, ERK1/2 and JNK in rat GMC treated with C5a.
Western blot was done to detect the phosphorylation levels of p38 MAPK (A and B), ERK1/2 (A and C) and JNK (A and D) in the GMC exposed to C5a (50 ng/ml) for different time points (0 min, 7.5 min, 15 min, 30 min and 60 min). Results were represented as means ± SD (n = 3 in each time point). Representative photographs were manifested. ** P<0.01 versus 0 min time point (non-treated).
Fig 6.
The effect of C5aR knockdown on the phosphorylation of p38 MAPK, ERK1/2 and JNK as well as the expression of IL-6 and TNF-α in C5a-induced GMC.
siC5aR was transfected into the GMC to silence C5aR gene followed by C5a stimulation at the dose of 50 ng/ml for different time points, and then above-mentioned molecules were examined by Western blot, RT-PCR and ELISA respectively. (A-D) Western blot was performed to detect the expression of C5aR and the phosphorylation levels of p38 MAPK (A and B), ERK1/2 (A and C) and JNK (A and D) in the GMC after C5a stimulation for 7.5 min (C and D) or 15 min (B). (E and F) RT-PCR was done to determine the mRNA levels of IL-6 and TNF-α in the GMC after C5a stimulation for 4 h. (G) ELISA was used to detect the release of IL-6 and TNF-α from the GMC after C5a stimulation for 8 h. Results were represented as means ± SD (n = 3 in each group). Representative photographs were shown. ** P<0.01 versus C5a group and siCTR + C5a group.
Fig 7.
The roles of p38 MAPK, ERK1/2 and JNK in the production of IL-6 and TNF-α in the GMC stimulated with C5a.
GMC were incubated with p38 MAPK inhibitor (SB203580, 10 μM), ERK1/2 inhibitor (U0126, 10 μM) and JNK inhibitor (SP600125, 10 μM) respectively for 30 min, and then stimulated with 50 ng/ml C5a for 4 h or 8 h. (A and B) RT-PCR was done to examine the mRNA levels of IL-6 and TNF-α in the GMC at 4 h. (C) ELISA was used to determine the release of IL-6 and TNF-α from the GMC at 8 h. Results were represented as means ± SD (n = 3 in each group). Representative photographs were exhibited. ** P<0.01 versus C5a group and DMSO + C5a group.