c, d TNF- (c) and iNOS (d) mRNA expression were determined with q-PCR

c, d TNF- (c) and iNOS (d) mRNA expression were determined with q-PCR. (BMDMs) were also used to demonstrate effect of PGRN on expressions and secretion of inflammatory cytokines induced by LPS. Results In RAW264.7 cells, rPGRN at concentrations below 80 ng/ml significantly promoted cell proliferation in dose dependent fashion. rPGRN significantly inhibited Cruzain-IN-1 LPS-induced change of phenotype (CD86/CD206 ratio) and function (tumor necrosis factor (TNF-) and inducible nitric oxide synthase (iNOS) expressions). LPS-stimulated secretion of TNF- and activated phosphorylation of IKK/, IB, p65, JNK and p38 and the nucleus translocation of NF-B p65 were also significantly downregulated by rPGRN. In addition, recombinant TNF- (rTNF-) significantly boosted TNF- and iNOS expression vs the control group. Moreover, anti-TNF- significantly inhibited LPS-induced TNF- and iNOS expression. In THP-1 and BMDM cells, reversing effect of rPGRN on LPS-enhanced expressions of TNF- and iNOS and secretion of TNF- was further demonstrated. Conclusions PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-B/MAPK signaling pathways. = 3, 0.05 (*), 0.001 (***) or 0.0001(****) Effects of rPGRN on proliferative capacity of RAW264.7 cells CCK-8 assays revealed that at concentrations below 80 ng/ml, rPGRN significantly promoted cell proliferation in dose dependent fashion after cultured for 24 and 48 h (Fig.?2a, b, d). Nevertheless, proliferative capacity of cells cultured at 160 and 320 ng/ml rPGRN approached to flat, and even declined. Furthermore, the proliferative capacity of RAW264.7 cells treated with different concentrations of Cruzain-IN-1 rPGRN was descending as processing time increases (Fig. ?(Fig.2a,2a, b, c, d). Open in a separate window Fig. 2 Effects of PGRN on RAW264.7 viability. a-c The effect of Cruzain-IN-1 5, 10, 20, 40, 80,160 and 320 ng/ml TSPAN9 rPGRN on RAW264.7 cells was detected by CCK-8 assay after treatment for 24 h (a), 48 h (b) and 72 h (c). The optical density was normalized to a relative value of 100% for untreated cells. d Cell proliferation curve. = 3, 0.05 (*), 0.01 (**) or 0.001 (***) Effects of rPGRN on LPS-induced M1 polarization in RAW264.7 cells RAW264.7 cells were stimulated by LPS plus 0, 5, 10, 20, 40 and 80 ng/ml rPGRN and normal medium containing equal amount of PBS to rPGRN and LPS groups was used as negative control. Their phenotype and function status were characterized by flow cytometry, q-PCR, Western blot and ELISA. The results showed that rPGRN at concentrations of 5, 10 and 20 ng/ml significantly reversed LPS-promoted CD86/CD206 expression ratio, of which 20 ng/ml rPGRN presented most obvious effect (Fig.?3a, b). Similarly, rPGRN significantly reversed LPS-enhanced mRNA of TNF- and iNOS at concentrations from 5 to Cruzain-IN-1 80 ng/ml, among which 10 ng/ml rPGRN presented most obvious effect (Fig. ?(Fig.3c,3c, d). It also inhibited LPS-enhanced protein expression of iNOS (at concentrations from 5 to 40 ng/ml) (Fig. ?(Fig.3e,3e, f) and TNF- (at 5 and 10 ng/ml) (Fig. ?(Fig.3e,3e, g), as well as secretion of TNF- (at 5 and 10 ng/ml) (Fig. ?(Fig.3h).3h). This implies that PGRN can inhibit LPS-induced M1 polarization in RAW264.7 cells. Open in a separate window Fig. 3 Inhibitory effects of rPGRN on CD86/CD206 ratio and inflammatory cytokines (TNF- and iNOS) expression in LPS-stimulate RAW264.7 cells. Cells were treated with LPS plus 5, 10, 20, 40, and 80 ng/ml rPGRN for 24 h and normal medium containing equal amount of PBS to rPGRN and LPS groups was used as negative control. a, b CD86/CD206 ratio was detected by flow cytometry. c, d TNF- (c) and iNOS (d) mRNA expression were driven with q-PCR. e-g TNF- (e, f) and iNOS (e, g) proteins expression had been revealed by Traditional western blot. h TNF- secretion appearance had been examined by ELISA. = 3, 0.05 (*), 0.01 (**), 0.001 (***) or 0.0001(****) The main element function of TNF- in LPS-induced macrophage M1 polarization Previous analysis provides identified that autocrine TNF- has a key function in apoptosis in LPS-induced macrophages [33]. It really is reported that PGRN exerts its anti-inflammatary actions mainly via binding also.