Kangfuxiaoyanshuan alleviates uterine inflammation and adhesion via inhibiting NF-κB p65 and TGF-β/MMP-2 signaling pathway in pelvic inflammatory disease rats

Abstract

Background and aims: Pelvic inflammatory disease (PID) is infection-induced inflammation of the female upper reproductive tract that results in high fever, ectopic pregnancy, infertility, and varying degrees of chronic pelvic pain. Recent clinical studies have shown that Kangfuxiaoyanshuan (KFXYS), a Traditional Chinese Medicine (TCM) formulation, may short the course of the disease and reduce the occurrence of PID sequelae, but its pharmacological action and potential mechanism have not been fully elucidated. Here, we aimed to investigate the therapeutic effects and mechanism of KFXYS in rats with PID. Materials and Methods: A PID rat model was constructed through endometrial mechanical injury and pathogen infection. The rectal temperature was measured during the 14-days course of treatment, and the white blood cell (WBC) count in the blood and the levels of cytokines (IFN-γ, IL-1β, IL-4, TNF-α) in the serum were evaluated by ELISA. Hematoxylin and eosin (HE) staining was performed to analyze pathological changes, and transmission electron microscopy (TEM) was used to observe ultrastructural changes. The p-p65/p65 protein expression was evaluated by western blotting and the levels of MMP-2 and TGF-β in adhesion tissues were assessed by immunohistochemistry. Results: KFXYS lowered the rectal temperature and the WBC counts in the blood in the acute stage of PID and alleviated inflammatory cell infiltration of the uterus, especially when combined with levofloxacin. KFXYS significantly decreased the levels of proinflammatory cytokines (IFN-γ, IL-1β, IL-4) and adhesion-related factors (TNF-α) and protected the ultrastructure of endometrial epithelial cells. Mechanistically, KFXYS inhibited the NF-κB activation by decreasing phosphorylation of p65, thus the alleviation of inflammation further reduced the expression of TGF-β and MMP-2, and inhibited the occurrence of uterine adhesions. Conclusion: These results revealed that KFXYS alleviated pelvic inflammation and effectively inhibits inflammation-associated adhesion, which indicated the potential role of KFXYS for treatment of PID and the prevention of PID sequelae.

Keywords: MMP-2; NF-κB; TGF-β; anti-inflammation; kangfuxiaoyanshuan; pelvic inflammatory disease (PID).

FIGURE 1

Schematic diagram of the experiment and changes in rat rectal temperature and WBC counts after treatment with different drugs. (A) Schematic diagram of the experimental setup. The first treatment day was denoted as D0.At D3, rats received 6 × 10⁸CFU E. coli and S. aureus solutions and underwent endometrial scratching. Treatment continued for 14 days, and tissues were sampled at D3, D14 and D49. (B) The changes in the rectal temperature of the rats during 14 days of treatment. The mean ± SEM are plotted in the graph; n = 4. (C) The infection indices and WBC counts determined from samples taken from the eyeballs of the rats during 14 days of treatment. The mean ± SEM are plotted in the graph; n = 7. Sham control: rats received laparotomy without manipulation and treatment. PID model: rats were untreated following the PID operation. Levofloxacin: rats that received 50 mg/kg/day levofloxacin orally for 14 days following the operation. KFXYS: rats that received 400mg/kg/day KFXYS rectally for 14 days following the operation. KFXYS + Levofloxacin: rats that received 400mg/kg/day KFXYS rectally and 50 mg/kg/day levofloxacin orally for 14 days following the operation.

FIGURE 2

Inflammatory changes in uterine tissue associated with drug treatment during the acute inflammation phase (D3). (A) Changes in inflammatory cells in the uterine mucosa were observed by HE staining. In the PID model group, the epithelial cells were hyperplastic and dense, showing papillary protrusion into the uterine cavity, some interstitial structures were destroyed, and a large number of chronic inflammatory cells were observed in the mucosal layer and muscle layer. In the Levofloxacin or KFXYS group, the formation of papillary protrusions by epithelial cell proliferation was reduced, and there was a small number of inflammatory cells and infiltrating neutrophils in the mucosal layer. There were almost no inflammatory cells and epithelial and glandular structures were intact in the KFXYS+Levofloxacin group. Images were taken at 50× or 100×; magnification. Scale bar: 100 μm. (B) Ultrastructural changes in the uterine mucosa of rats observed by TEM. Intact microvilli (black arrows), intercellular connections (red arrows), secretory vesicles (purple arrows), and lipid droplets (green arrows) were observed. N, nucleus; M, mitochondrion; A, autolysosome. Images were taken at 5000× or 30000× magnification.

FIGURE 3

Decreasing effect of different drugs on the blood levels of the immune-related factors IL-1β, IFN-γ, IL-4, and TNF-α in rats. Expression changes are presented as the mean ± SD (n = 4). The data were analyzed using one-way ANOVA and unpaired t test. *p < 0.05 vs the PID model group, **p < 0.01 vs the PID model group, and *** p < 0.001 vs the PID model group.

FIGURE 4

Histopathological and ultrastructural changes in uterine tissue after 14 days of treatment and the expression of p65 and p-p65. (A) Uterine mucosa adhesion was observed by HE staining. The black arrows indicate bands of adhesive tissue. Images were taken at 50× or 100× magnification. Scale bar: 200 μm. (B) Ultrastructural changes in the uterine mucosa of rats observed by TEM. Intact microvilli (black arrows), intercellular connections (red arrows), secretory vesicles (purple arrows), and lipid droplets (green arrows) were observed. N, nucleus; M, mitochondrion; A, autolysosome. Images were taken at 5000× or 30000× magnifications. Scale bar: 1 μm. (C) Western blot analysis was used to measure the expression levels of p65 and p-p65 at D14.β-actin was used as an internal control. (D) The ratio of p-p65/p65 protein levels from three independent western blot analyses.

FIGURE 5

The expression of TGF-β at D14 and D49 by immunohistochemistry analysis. (A) Immunohistochemistry was used to test the protein expression of TGF-β in adhesive uterine tissue at D14. (B) Semiquantitative analysis by the H-score of TGF-β at D14. (C) The protein expression of TGF-β in adhesive uterine tissue at D49. (D) Semiquantitative analysis by the H-score of TGF-β at D49 Scale bar: 100 μm. **p < 0.01 vs the PID model group, and *** p < 0.001 vs the PID model group.

FIGURE 6

Histopathological images showing the degree of adhesion of uterine tissue at D49 and analysis of MMP-2 expression by immunohistochemistry. (A) Uterine mucosa adhesion was observed by HE staining. The black arrows indicate bands of adhesive tissue. Images were taken at 50× or 100× magnification. (B) Immunohistochemistry was used to analyze the protein expression of MMP-2 in adhesive uterine tissue from the different groups. (C) Semiquantitative analysis by the H-score of MMP-2. (D) Western blot analysis was used to measure the expression levels of MMP-2 at D49. β-actin was used as an internal control. Scale bar: 200 μm. **p < 0.01 vs the PID model group, and *** p < 0.001 vs the PID model group.