LPS Induces Preeclampsia-Like Phenotype in Rats and HTR8/SVneo Cells Dysfunction Through TLR4/p38 MAPK Pathway

Abstract

Accumulating evidence has shown that preeclampsia (PE) was associated with an aberrant maternal-fetal inflammatory response. In the present study, we first found that in human PE placentas levels of toll-like receptor 4 (TLR4), phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) and inflammatory cytokines IL-6 and MCP-1 were significantly upregulated. Next, we demonstrated a notable increase in systolic blood pressure (SBP) and proteinuria in lipopolysaccharide (LPS)-treated pregnant rats and concomitant high levels of TLR4 and p-p38 in these PE-like rat placentas, which led to aberrant overexpression of both IL-6 and MCP-1, as well as deficient trophoblast invasion and spiral artery (SA) remodeling, and these abnormalities were ameliorated by SB203580, a reported inhibitor of p38. In vitro we further confirmed that LPS triggered the activation of TLR4/p38 signaling pathway, which promoted trophoblast apoptosis and damaged trophoblastic invasion via downstream effectors IL-6 and MCP-1; these mutations were rectified by silencing this signaling pathway. These findings elaborated potential mechanisms that aberrant TLR4/p38 signaling might contribute to PE and LPS-induced PE-like symptom by damaging trophoblast invasion and SA remodeling via activating inflammatory cytokines including IL-6 and MCP-1.

Keywords: inflammation; lipopolysaccharide; p38 MAPK; preeclampsia; spiral artery; toll-like receptor 4.

Figure 1

Inflammatory cytokines increased in the placenta of PE-bearing women with high TLR4/p38 levels. (A) Immunohistochemistry showed that TLR4 protein staining substantially enhanced in the cytoplasm of PE placenta trophoblasts in comparison with that of normal pregnancy (NP). (B,C) Western blot illustrated that the expression levels of TLR4 and p-p38 were markedly increased in the placentas of PE compared with that found during normal pregnancy. (D,E) Western blot and real-time PCR showed that both IL-6 and MCP-1 in PE placentas were significantly higher than those of normal pregnancy on protein and mRNA levels. Values denote the mean ± s.d.; ^*p < 0.01 vs. normal pregnancy. NP, normal pregnancy; PE, preeclampsia.

Figure 2

Expressions of TLR4 and p-p38 and their effects on trophoblast invasion and SA remodeling in LPS-treated rats. (A) Immunohistochemistry suggested that TLR4 staining was enhanced in the cytoplasm in LPS-treated and LPS plus p38 inhibitor SB203580-treated rats, which was predominantly expressed by spongiotrophoblast cell, trophoblastic giant cell, and glycogen cell in the basal zone as well as trophoblastic epithelium in the labyrinth of rat placenta. Scale bar = 100 μm. (B) Western blot revealed that LPS treatment gave rise to TLR4 expression and p38 phosphorylation in the placenta of pregnant rats. The addition of SB203580 dramatically abrogated p38 activation, whereas showed little alterations on TLR4 expression. Values denote the mean ± s.d.; ^*p < 0.01 vs. control, ^#p < 0.01 vs. LPS-treated group. (C) SAs from all groups exhibited evidence of remodeling, including the endovascular trophoblast cells of cytokeratin staining (arrows) resting on a fibrinoid layer of PAS staining (arrows) and the smooth muscle cells of α-SMA staining (arrows). Scale bar = 100 μm. (D) The percentages of trophoblast, fibrinoid, and VSMC of total SA contour length were determined. Values denote the mean ± s.d.; ^*p < 0.01 vs. control, ^#p < 0.05 vs. LPS-treated group. Ctrl, control group; SB, SB203580.

Figure 3

Effects of p38 inhibition on the hypertension and proteinuria of LPS induced PE-like rats and the morphological features of their kidneys. (A,B) The SBP and urinary protein were presented, respectively. No remarkable alterations in SBP and 24 h urinary protein levels were observed before LPS infusion among different groups. After injection, SBP levels of LPS-treated rats were much higher and were prohibited and restored by SB203580 from GD 17. The 24 h urinary albumin excretion was significantly higher in LPS-treated group after LPS infusion, and these abnormalities were essentially prevented by treatment of SB203580. Values denote the mean ± s.d.; p > 0.05 vs. control before LPS infusion, ^*p < 0.05, ^**p < 0.01 vs. control on the corresponding GD after LPS infusion. ^##p < 0.01 vs. LPS-treated group on the corresponding GD after LPS infusion. (C) Kidney ultrastructure on TEM in different groups. GBM thickening and foot process effacement were demonstrated in LPS group, which were attenuated in LPS-p38 inhibitor group. Scale bar = 1 μm. (D) Mesangial and glomerular area by PAS staining. There occurred moderate mesangial proliferation, GBM thickening, and adhesion of the glomerular tuft to the Bowman’s capsule in LPS-treated group, indicative of advanced renal injury. These mutations were attenuated in LPS-p38 inhibitor group. Values denote the mean ± s.d.; ^*p < 0.01 vs. control, ^#p < 0.01 vs. LPS-treated group. Scale bar = 100 μm. Ctrl, control group; SB, SB203580.

Figure 4

Alterations of inflammatory cytokines expression in the placenta and kidney of different pregnant groups in rats. Western blot and real-time PCR presented that LPS treatment gave rise to the expressions of inflammatory markers IL-6 and MCP-1 on both protein and mRNA levels in (A,B) placentas and (C,D) kidney cortex; aberrant expressions of these two proteins and mRNA were abolished by pretreatment of SB203580. Values denote the mean ± s.d.; ^*p < 0.01 vs. control, ^#p < 0.05 vs. LPS-treated group. Ctrl, control group; SB, SB203580.

Figure 5

Determination of expression of TLR4, p-p38, and inflammation cytokines and their interplays in cultured HTR-8/SVneo cell. (A,B) Both western blot and real-time PCR demonstrated a relatively high knockdown efficiency of TLR4 or p38 gene after shRNA transfection for 24 h. Values denote the mean ± s.d.; ^*p < 0.01 vs. control. (C,D) Western blot demonstrated that LPS treatment promoted TLR4 expression and p38 phosphorylation compared with controls. However, in comparison with LPS group, expressions of TLR4 and p-p38 in LPS-treated cells were significantly decreased by transfection with TLR4 shRNA; p38 shRNA transfection in LPS-treated cells dramatically deleted p38 activation without affecting TLR4 expression. Values denote the mean ± s.d.; ^*p < 0.01 vs. control, ^#p < 0.01 vs. LPS-treated group. (E,F) Western blot and real-time PCR showed that excessive expressions of IL-6 and MCP-1 were induced on both protein and mRNA levels in LPS-treated cells compared with control; these aberrant expressions were abolished by TLR4 or p38 gene knockdown. Values denote the mean ± s.d.; ^*p < 0.01 vs. control and ^#p < 0.01 vs. LPS-treated group. (G) Immunofluorescence staining revealed TLR4 expression in the cytoplasm of the HTR-8/SVneo cells. Compared with control, LPS treatment markedly enhanced TLR4 staining, which was decreased by transfection with TLR4 shRNA. Nevertheless, there were no distinguishable differences on TLR4 staining between p38 shRNA transfection and LPS treatment. Scale bar = 20 μm. (H) CO-IP demonstrated the interactions between TLR4 and p38. Cell lysates were immunoprecipitated with anti-TLR4 antibodies and were subjected to immunoblot with the indicated antibodies. Ctrl, control group; Scramble, scrambled shRNA.

Figure 6

Determining invasion and apoptosis of LPS-treated HTR-8/SVneo cells in different groups. (A) Transwell Matrigel invasion assay analyzed HTR-8/SVneo cells invasion, and the results were represented as invasion percentage. In comparison with control, the invasive ability of LPS-treated cells decreased, which was ameliorated by transfection with TLR4 shRNA or p38 shRNA. Values denote the mean ± s.d.; ^*p < 0.01 vs. control, ^#p < 0.01 vs. LPS-treated group, scale bar = 40 μm. (B) The total apoptotic rates in different groups were identified. After LPS treatment for 24 h, the total apoptotic rate was enhanced in HTR-8/SVneo cells, but was reversed by either TLR4 or p38 gene knockdown. Ctrl, control group; Scramble, scrambled shRNA.