Ginsenoside Rb1 Mitigates Escherichia coli Lipopolysaccharide-Induced Endometritis through TLR4-Mediated NF-κB Pathway

Abstract

Endometritis is the inflammatory response of the endometrial lining of the uterus and is associated with low conception rates, early embryonic mortality, and prolonged inter-calving intervals, and thus poses huge economic losses to the dairy industry worldwide. Ginsenoside Rb1 (GnRb1) is a natural compound obtained from the roots of Panax ginseng, having several pharmacological and biological properties. However, the anti-inflammatory properties of GnRb1 in lipopolysaccharide (LPS)-challenged endometritis through the TLR4-mediated NF-κB signaling pathway has not yet been researched. This study was planned to evaluate the mechanisms of how GnRb1 rescues LPS-induced endometritis. In the present research, histopathological findings revealed that GnRb1 ameliorated LPS-triggered uterine injury. The ELISA and RT-qPCR assay findings indicated that GnRb1 suppressed the expression level of pro-inflammatory molecules (TNF-α, IL-1β and IL-6) and boosted the level of anti-inflammatory (IL-10) cytokine. Furthermore, the molecular study suggested that GnRb1 attenuated TLR4-mediated NF-κB signaling. The results demonstrated the therapeutic efficacy of GnRb1 in the mouse model of LPS-triggered endometritis via the inhibition of the TLR4-associated NF-κB pathway. Taken together, this study provides a baseline for the protective effect of GnRb1 to treat endometritis in both humans and animals.

Keywords: Ginsenoside Rb1; NF-κB pathway; TLR4; endometritis; lipopolysaccharide.

Figure 1

(A) The structure of Ginsenoside Rb1. (B) Animal treatment protocol of this study.

Figure 2

Effects of GnRb1 against LPS-triggered uterine injury. (A) The morphology of uterine tissue. (B) The control group (CG). (C) The LPS group. (D,E) The LPS + GnRb1 (25 and 50 mg/kg, respectively). (F) Histopathological score of uterine sections. The scale bar is of 100 µm (200× magnification). CG represents the control group. LPS reveals the LPS-induced groups. The 25 and 50 are the GnRb1-administrated groups representing 25 mg/kg and 50 mg/kg per animal, respectively. GnRb1 indicates the Ginsenoside Rb1. Data statistics are demonstrated as mean ± S.E.M. The # p < 0.001 CG versus LPS group, *** p < 0.001 and ns indicates the nonsignificant difference between CG versus LPS + GnRb1 groups.

Figure 3

Consequence of GnRb1 against MPO activity assay and W/D ratio in mice uterus. (A) MPO (U/G) activity. (B) W/D ratio. CG represents the control group. LPS reveals the LPS-induced groups. The 25 and 50 are the GnRb1-administrated groups representing 25 mg/kg and 50 mg/kg per animal, respectively. GnRb1 indicates the Ginsenoside Rb1. MPO indicates the myeloperoxidase, whereas W/D depicts the wet to dry weight ratio. Data statistics are demonstrated as mean ± S.E.M. The # p < 0.001 CG versus LPS group, * p < 0.05, ** p < 0.01, and ns indicates the nonsignificant difference between CG versus LPS + GnRb1 groups.

Figure 4

Effects of GnRb1 against LPS-induced production of pro-inflammatory cytokines. The concentration of: (A) the TNF-α (pg/mL); (B) the IL-1β (pg/mL); and (C) IL-6 (pg/mL). The relative mRNA expression levels of (D) TNF-α, (E) IL-1β; and (F) IL-6. CG represents the control group. LPS reveals the LPS-induced groups. The 25 and 50 are the GnRb1-administrated groups representing 25 mg/kg and 50 mg/kg per animal, respectively. GnRb1 indicates the Ginsenoside Rb1. Data statistics are demonstrated as mean ± S.E.M. The # p < 0.001 CG versus LPS group, * p < 0.05, ** p < 0.01, *** p < 0.001, and ns indicates the nonsignificant difference between CG versus LPS + GnRb1 groups.

Figure 5

Effects of GnRb1 against LPS-induced production IL-10 concentration. The concentration of (A) IL-10 (pg/mL). The relative mRNA expression level of (B) IL-10 in uterine tissue. CG represents the control group. LPS reveals the LPS-induced groups. The 25 and 50 are the GnRb1-administrated groups representing 25 mg/kg and 50 mg/kg per animal, respectively. GnRb1 indicates the Ginsenoside Rb1. Data statistics are demonstrated as mean ± S.E.M. The ** p < 0.001 CG versus LPS group, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns indicates the nonsignificant difference between CG versus LPS + GnRb1 groups.

Figure 6

Effects of GnRb1 against LPS-induced expression of the TLR4. (A) The relative mRNA expression level of the TLR4 gene. (B) The protein expression of TLR4. CG represents the control group. LPS reveals the LPS-induced groups. The 25 and 50 are the GnRb1-administrated groups representing 25 mg/kg and 50 mg/kg per animal, respectively. GnRb1 indicates the Ginsenoside Rb1. Data statistics are demonstrated as mean ± S.E.M. The # p < 0.001 CG versus LPS group, ** p < 0.01, *** p < 0.001, and ns indicates the nonsignificant difference between CG versus LPS + GnRb1 groups.

Figure 6

Effects of GnRb1 against LPS-induced expression of the TLR4. (A) The relative mRNA expression level of the TLR4 gene. (B) The protein expression of TLR4. CG represents the control group. LPS reveals the LPS-induced groups. The 25 and 50 are the GnRb1-administrated groups representing 25 mg/kg and 50 mg/kg per animal, respectively. GnRb1 indicates the Ginsenoside Rb1. Data statistics are demonstrated as mean ± S.E.M. The # p < 0.001 CG versus LPS group, ** p < 0.01, *** p < 0.001, and ns indicates the nonsignificant difference between CG versus LPS + GnRb1 groups.

Figure 7

Effects of GnRb1 against LPS-induced expression NF-κB pathway proteins measured by ELISA. (A) The expression levels of the NF-κB p65 and its phosphorylated (p-NF-κB p65) form. (B) The expression levels of total IκBα and its phosphorylated (p-IκBα) form. CG represents the control group. LPS reveals the LPS-induced groups. The 25 and 50 are the GnRb1-administrated groups representing 25 mg/kg and 50 mg/kg per animal, respectively. GnRb1 indicates the Ginsenoside Rb1. Data statistics are demonstrated as mean ± S.E.M. The # p < 0.001 CG versus LPS group, ** p < 0.01, and ns indicates the nonsignificant difference between CG versus LPS + GnRb1 groups.