Table 1.
Primers of human IL-1β, IL-6, TLR4 and β-actin.
Fig 1.
Effects of RGZ on the expression of inflammatory cytokine in the uterine tissue.
Compared with the DMSO group, the expression of IL-1β and IL-6 was significantly elevated in the LPS group and LPS+RGZ group. RGZ significantly reduced the expression of IL-1β and IL-6 in LPS-treated animals. The values presented are the mean ± SEM (n = 10 in each group). ##P < 0.01 vs. DMSO group. *P < 0.05, vs. LPS group.
Fig 2.
The effect of RGZ on uterus tissue pathologic changes during LPS-induced endometritis for H&E staining.
Scale bar: 100 μm. Mice were given an intraperitoneal injection of RGZ (10 mg/kg) 2 hours prior to uterine perfusion of LPS. Uterus tissue was collected 24 h following LPS challenge. a DMSO control group, b RGZ control group, c LPS group, d LPS + RGZ group. Neutrophils were indicated by the arrows. (e) Inflammation score of uterus tissue. The histopathologic scoring was a cumulative score of hyperemia and neutrophils infiltration. The values presented were the means ± SEM (n = 10). ns, not significant, #P < 0.05 and *P < 0.05 compared with DMSO control group and LPS group, respectively.
Table 2.
The histopathologic scoring criteria.
Fig 3.
Immunohistochemical assessment of TLR4 expression in endometrial tissue of mouse.
a DMSO control group, b RGZ control group, c LPS group, d LPS + RGZ group. TLR4 staining levels in endometrial tissues showed a reduced intensity in LPS + RGZ group compared to the LPS group. TLR4 staining levels in endometrial tissue indicated a reduced intensity in the RGZ group compared to the DMSO control group. The values presented were the means ± SEM (n = 10). ns, not significant, #P < 0.05 and *P < 0.05 compared with DMSO control group and LPS group, respectively.
Fig 4.
The effects of RGZ on LPS-induced activation of TLR4 and NF-κB in vivo.
The protein levels of TLR4, p-p65 and p-IκBα in uterus tissues were measured by Western blotting. Data were represented the mean ± SEM (n = 10). #P < 0.05 and *P < 0.05 compared with DMSO control group and LPS group, respectively.
Fig 5.
The effects of RGZ on the cell viability of HESCs.
Cells were treated with the indicated concentration of RGZ (0~40 μM/L) for 24 h, and the cell viability was assessed by CCK-8 kits. #P < 0.05 compared with control group.
Fig 6.
mRNA levels of cytokines and TLR4 expression from HESCs stimulated by LPS and RGZ.
a-b mRNA levels of IL-1β and IL-6 in LPS-stimulated HESCs. GAPDH was used as a control. Control is Effects of RGZ on cytokines and TLR4 expression. the control group; LPS (0.01,0.05,0.25,1,5μg/mL) are the LPS groups. c-e Control is the control group; LPS (1μg/mL) is the LPS group; and 10 and 20 are the RGZ treatment groups representing 10 μM/L and 20 μM/L per cell plate, respectively. The data presented were the means ± SEM. ##P < 0.01, *P < 0.05 and **P < 0.01 compared with control group and LPS group, respectively.
Fig 7.
The effects of RGZ and TAK-242 on LPS-induced activation of TLR4 and NF-κB in vitro.
The protein levels of TLR4, p-p65 and p-IκBα in HESCs were measured by Western blotting. GAPDH served as the control. Control is the control group; LPS is the LPS group; TAK-242 was the 1 μM/L TAK-242 treatment groups per cell plate, RGZ was the 10 μM/L RGZ treatment groups per cell plate, LPS+RGZ was the 30min incubation of the cell pretreatment with 10 μM RGZ followed by LPS induced for 24 h incubation, LPS+RGZ+TAK-242 was the 2 h incubation of 1 μM TAK-242 combined with 30min incubation of with 10 μM RGZ followed by LPS induced for 24 h incubation. Data represent the mean ± SEM. ##P < 0.01, *P < 0.05 and **P < 0.01 compared with control group and LPS group, respectively.
Fig 8.
RGZ inhibit NF-κB p65 translocation into the nucleus and expression of TLR4 in vitro.
Translocation of the NF-κB p65 subunit from the cytoplasm into the nucleus was assessed by immunofluorescence staining. After LPS stimulation, TLR4 was markedly increased in uterus tissue compared to the control group. Treatment with RGZ and RGZ+TAK-242 reduced the expression of TLR4. Scale bar: 50 μm. Blue spots represent cell nuclei, and green spots indicate TLR4 staining. red spots indicate p-p65 staining.
Fig 9.
Schematic diagram of signaling pathways related to the anti-inflammatory effects of RGZ on LPS-induced inflammation.