Oxidative Stress Mediates the Dual Regulatory Effects of Bovine Uterine ECM Remodeling Through the TGF-β1/Smad3 Pathway: Molecular Mechanisms of MMPs and COL-IV Imbalances

Abstract

Bovine endometritis is a common endocrine and reproductive disorder in postpartum dairy cows, closely associated with elevated systemic oxidative stress. This disease can lead to delayed uterine involution, repeated breeding failure, and significant economic losses in the dairy industry. Studies suggest that oxidative stress may contribute to the pathological progression of endometritis by regulating ECM remodeling, but the specific molecular mechanisms remain unclear. ECM homeostasis relies on the coordinated action of matrix metalloproteinases (e.g., MMP2, MMP9) and collagen (e.g., type IV collagen, COL-IV), while the TGFβ1/Smad3 signaling pathway is implicated in ECM metabolic regulation. Therefore, elucidating the regulatory mechanisms of oxidative-stress-mediated TGFβ1/Smad3 signaling on ECM remodeling is crucial for understanding the pathogenesis of endometritis. This study investigates postpartum bovine uterine tissues, comparing inflammatory cytokines (IL-1β, IL-6, TNF-α) and oxidative-stress-related factors (GPx, SOD, CAT) between healthy and endometritis groups. Additionally, the differences in ECM-remodeling-associated proteins (MMP2, MMP9, COL-IV) and TGFβ1/Smad3 pathway activity are analyzed. To further validate the mechanisms, an oxidative stress model is established in vitro by treating bovine endometrial epithelial cells (bEECs) with 200 μM H₂O₂ for 4 h, followed by the valuation of the same indicators. Furthermore, gene silencing to downregulate Smad3 expression or inhibitor-mediated suppression of TGFβ1/Smad3 pathway activity is performed to observe their regulatory effects on MMP2, MMP9, and COL-IV. The results demonstrate that oxidative-stress-mediated endometritis significantly upregulates MMP2, MMP9, and the TGFβ1/Smad3 pathway activity, while suppressing COL-IV expression. Functional genetic experiments further reveal the dual regulatory role of the TGFβ1/Smad3 pathway in ECM remodeling: (1) pathway activation promotes MMP2/MMP9 expression, accelerating COL-IV degradation; (2) Smad3 positively regulates COL-IV synthesis. These findings provide a theoretical basis for targeting the TGFβ1/Smad3 pathway to mitigate the pathological progression of endometritis.

Keywords: ECM remodeling; MMPs; TGF-β1/Smad3 signaling pathway; bovine endometritis; oxidative stress.

Figure 1

Detection of inflammatory factors and antioxidant-related enzymes in the healthy and endometritis groups of dairy cows. (A) Histopathological sections of the healthy group and subclinical group. (B–D) RT-qPCR analysis of IL-1β, IL-6, and TNF-α mRNA expressions. (E–G) RT-qPCR analysis of the relative mRNA expression levels of CAT, GPx1, and SOD in bovine uterine tissues. E denotes endometrial epithelial cells, G denotes granulocytes, and L indicates lymphocytes. Scale bar = 100 μm. RT-qPCR data were analyzed using t-tests compared to the control group. Significant differences are marked as * p < 0.05, ** p < 0.01.

Figure 2

Detection of TGF-β1/Smad3 signaling pathway and ECM-remodeling-related protein expression in the healthy and endometritis groups of dairy cows. (A) Immunohistochemical (IHC) analysis of COL-IV protein expression in the endometrial epithelium. (B,C) Relative expression level of TGF-β1 to β-actin. (D–F) WB analysis of relative expression level of MMP2 and MMP9 to β-actin. E denotes endometrial epithelial cells, G represents granulocytes, and L indicates lymphocytes. Scale bar = 100 μm. Experiments were repeated three times. Data were analyzed using t-tests compared to the control group. Significant differences are marked as * p < 0.05, ** p < 0.01. Original western blots are presented in File S1.

Figure 3

Activation of inflammation and oxidative stress in bEECs by H₂O₂. (A) CCK-8 assay for cell viability. (B) Relative ROS levels. (C–E) Changes in mRNA expressions of IL-1β, IL-6, and IL-8. Scale bar = 100 μm. Data were analyzed using t-tests compared to the control group. Significant differences are marked as * p < 0.05 ** p < 0.01.

Figure 4

Activation of TGFβ1/Smad3 pathway by H₂O₂. (A) RT-qPCR analysis of TGFβ1 mRNA expression. (B,C) RT-qPCR and Western blot analysis of Smad3 expression. (D) Relative expression level of P-Smad3 to β-actin (E) Immunofluorescence detection of nuclear P-Smad3 protein localization. Scale bar = 20 μm. Data were analyzed using t-tests compared to control group. Significant differences are marked as * p < 0.05. Original western blots are presented in File S1.

Figure 5

Activation of ECM remodeling by H₂O₂. (A) Relative expression level of MMP2 to β-actin. (B,C) RT-qPCR and Western blot analysis of MMP9 expression. (D,E) RT-qPCR and Western blot analysis of COL-IV expression. Data were analyzed using t-tests compared to control group. Significant differences are marked as * p < 0.05, ** p < 0.01. Original western blots are presented in File S1.

Figure 6

Effects of TGFβ1/SMAD3 pathway expression on ECM-remodeling-related protein levels. (A–C) Western blot (WB) analysis of relative expression of Smad3 and phosphorylated Smad3 (P-Smad3). (D) Immunohistochemical (IHC) staining showing nuclear localization of P-Smad3. (E–G) WB analysis of relative expressions of MMP2, MMP9, and COL-IV. (H–J) RT-qPCR analysis of relative mRNA expressions of Smad3, MMP9 (H), and COL-IV. Scale bar = 20 μm. Data were analyzed using one-way ANOVA compared to control group. Different lowercase letters indicate statistically significant differences (p < 0.05). Original western blots are presented in File S1.