Smad signaling coincides with epithelial-mesenchymal transition in a rat model of intrauterine adhesion

Abstract

Purpose: Intrauterine adhesion (IUA) is a fibrotic disease mainly caused by tissue injury, yet the mechanism is poorly understood. The aim of this study was to investigate the roles of TGF-β1/BMP7/Smad signaling coincident with epithelial-mesenchymal transition (EMT) in IUA.

Methods: Twenty-four female SD rats were divided into IUA and sham groups. For each animal, a mechanical injury or sham operation was performed on the left uterus (IUA-L, Sham-L), and the right uterus (IUA-R, Sham-R) was used as the control. Animals were sacrificed in batches on days 7 and 28. The endometrial morphology, number of endometrial glands, microvascular density (MVD), area of endometrial fibrosis and immunohistochemistry (IHC) analysis of biomarkers of EMT, as well as levels of TGF-β1, phosphorylated Smad3 (pSmad3), BMP7, phosphorylated Smad1/5 (pSmad1/5) and estrogen receptor (ER) were evaluated. Besides, the correlation between these IHC markers was also analyzed. RT-PCR and western blot were used to test relevant genes.

Results: Compared with other groups, the IUA-L group showed a significant decrease in the number of glands and MVD. And it also showed a significant increase in the stromal fibrosis rate and a-SMA level. Moreover, in the IUA-L group, TGF-β1 and pSmad3 levels were consistently high, and levels of BMP7, pSmad1/5 and ER were low. EMT markers E-cadherin was decreased, while N-cadherin was increased. Sham and control groups showed no significant difference in these markers. In addition, E-cadherin with a-SMA, fibrosis rate with BMP7, TGF-β1 with pSmad3 and BMP7 with pSmad1/5 showed correlation in IUA-L group, which had statistical significance. The mRNA expression of TGF-β1, a-SMA and ccn2 in 7 d IUA-L was higher than 7 d IUA-R while BMP7 was lower, which had significant difference. The protein expression of BMP7 in 7 d IUA-L was lower than 7 d IUA-R, which had significant difference.

Conclusions: These results suggest a potential role of Smad signaling together with EMT in endometrial fibrosis development.

Keywords: BMP7; EMT; IUA; Smad; TGF-β1; fibrosis.

Figure 1

Mechanical surgery to establish an IUA rat model. A. Normal rat uterine appearance. The uterine had two horns and the color appeared red. B. After disinfection, tweezers were used to pick Y-shaped uterus, the endometrial surface was damaged by mechanical injury. Using scissors to cut endometrium layer carefully, kept the depth to superficial muscle layer. Sterile gauzes were needed to make the surgical area clean. C. Suture after the operation. The injured uterus looked distorted and unshapely. D. The profile of rat uteri on day 7, hydrops was found in IUA-L uterus group as the Sham-L group showed no difference with IUA-R and Sham-R group. E. IUA-L uterus was blocking and IUA-R uterus enjoyed a normal uterine cavity.

Figure 2

The micro view of an IUA rat model. A. HE staining of rat uteri after 7 days; the IUA-L uterine cavity totally disappeared and gland quantity decreased sharply. Neutrophil cells and red blood cells also filled in IUA-L endometrium. Scale bar = 200 µm. B. Immunohistochemical staining of the endometrium for CD31. Light yellow circle symbolized as active capillary, MVD was counted by CD31. Scale bar = 20 µm. C. Gland quantity decreased significantly in the 7 d and 28 d IUA-L group compared with that of other groups, P<0.05. Other groups showed no differences. D. MVD decreased significantly in the 7 d and 28 d IUA-L group compared with that of other groups, P<0.01. Other groups showed no differences.

Figure 3

The IUA rat model became fibrotic. A. Typical a-SMA green staining of IUA-L stroma on day 7, IUA-R, Sham-L and Sham-R endometrium did not show positive immunofluorescence staining. a-SMA was not normally expressed in endometrial layer. But in IUA-L group, a-SMA was seen in the fibrous stroma. Here, we provided a confocal image of a-SMA for a better view but still quantified a-SMA by IHC. Scale bar = 25 µm. B. Masson staining of the endometrium 7 days after surgery in the IUA and sham groups. The extent of blue color in endometrial layer meant deeper fibrosis rate, as we can see, IUA-L group showed much deeper blue than IUA-R, Sham-L and Sham-R group. Besides, less glands and epithelial cells were found in IUA-L group. Scale bar = 20 µm. C. IHC staining of a-SMA in the IUA-L group was highest among the groups, P<0.01. According to Masson staining, the average fibrosis rate was 57.17%±0.2658 and 45.81%±0.1209 in IUA-L on day 7 and day 28, P<0.01, respectively. D. The mRNA expression of ccn2 and a-SMA. ccn2 and a-SMA were relevant fibrosis markers. The mRNA level of ccn2 in 7 d IUA-L group was 25 folds meaningfully higher than 7 d IUA-R group. Even though the mRNA expression of a-SMA showed no difference between 7 d IUA-L and IUA-R group, we could still observe the potential higher expression. As for Sham-L and Sham-R group, mRNA expression of ccn2 and a-SMA both showed no significant difference.

Figure 4

Representative immunostaining of TGF-β1 and pSmad3 in rat model on day 7. A. TGF-β1 staining was increased progressively in the epithelial cell cytoplasm in the IUA-L uterus but was negative in IUA-R and sham uteri. Scale bar = 20 µm. B. pSmad3 staining was positive in the nuclei of epithelial cells and a small number of stromal cells in the IUA-L group but negative in the normal endometrium too. The location and expression of TGF-β1 and pSmad3 were identical in IUA-L group. Scale bar = 20 µm. C. The P value of the differences in TGF-β1 was P<0.01 both on day 7 and was P<0.05 on day 28. D. The P values of the differences in pSmad3 on day 7 and day 28 were P<0.01. Scale bar = 20 µm. E. The gene level of TGF-β1 was more 10 folds higher in 7IL compared with 7IR group (P = 0.0390). This was consistent with immunostaining of TGF-β1.

Figure 5

Representative immunostaining of BMP7 and pSmad1/5 in rat model on day 7. A. BMP7 was fully expressed in the stromal cell cytoplasm. The immunostaining intensity of BMP7 in IUA-L decreased, it was still strong in IUA-R and sham endometrium. B. pSmad1/5 was also located in the nuclei of epithelial and stromal cells. And it showed less positive in IUA-L endometrium when compared with IUA-R, Sham-L and Sham-R. BMP7 and pSmad1/5 were similarly decreased in the IUA-L group but maintained high expression in the other groups. C, D. The MOD of BMP7 and pSmad1/5 were lower than other groups. The P value of the differences in BMP7 and pSmad1/5 was P<0.05 on day 7 and day 28. Scale bar = 20 µm.

Figure 6

The mRNA and protein expression of BMP7. A. 6 samples were collected from each group. Western blot membrane from left to right represented 7IL, 7IR, 7SL and 7SR. The first row was BMP7 protein marker, and the second row was their GAPDH. B. T-test analysis proved BMP7 protein expression was lower in 7IL compared with 7IR (P = 0.0286). As 7SL showed no significant difference with 7SR. C. The mRNA expression of BMP7 was significantly decreased in 7IL compared with 7IR (P = 0.0214). While 7SL and 7SR had no statistical difference.

Figure 7

EMT markers changed in the IUA-L group. A. Usually, E-cadherin affluently displayed in normal epithelial cells. After the mechanical injury, it was hard to detect E-cadherin staining in IUA-L. Scale bar = 20 µm. B. N-cadherin was an epithelial marker but rarely expressed in epithelial cells. This study found strong N-cadherin staining in IUA-L epithelial cells but light staining in normal epithelial cells. Scale bar = 20 µm. C. The MOD level of E-cadherin after day 7 and day 28 declined significantly in the IUA-L group, P<0.01. D. N-cadherin level after day 7 and day 28 increased significantly in the IUA-L group compared with that of other groups, P<0.01.

Figure 8

Representative Vimentin and ER immunostaining. A. Vimentin was a mensenchymal marker normally expressed in the cytoplasm of stromal cells and stayed negative in epithelial cells. Here, we could found vimentin not only located in the cytoplasm of both epithelial cells and stromal cells in IUA-L group. Moreover, the morphology of the glands in IUA-L group also became stiffened, these epithelial cells looked much similar as stromal cells. Scale bar = 25 µm. B. ER was mainly located in the nuclei of stromal cells. After the mechanical injury, ER expression decreased in IUA-L group. C. ER level after day 7 and day 28 decreased significantly in the IUA-L group compared with that of other groups, P<0.01.