Essential role of ATG7 in LPS-induced endometrial inflammatory injury in goats

Abstract

The economic losses caused by uterine infections in ruminants have received increasing attention. Autophagy-related 7 (ATG7) has been demonstrated to be capable of regulating apoptosis, but surprisingly, ATG7 can both promote and inhibit apoptosis in different cellular contexts. However, the role of ATG7 in endometrial cell apoptosis during the postpartum period remains unclear. Herein, the location and expression of ATG7 was determined after mice were treated with lipopolysaccharide (LPS) in the uterus. The results showed that lipopolysaccharide (LPS) increased ATG7 expression in endometrial epithelial cells (EECs) but not endometrial stromal cells. The apoptosis of goat EECs was increased under LPS treatment, and LPS further led to an increase in bax expression and a decline in bcl-2 expression in goat EECs. Silencing of ATG7 inhibited goat EEC apoptosis with LPS treatment. The role of ATG7 in the regulation of goat EEC apoptosis was further confirmed by overexpression. ATG7 may serve as an essential regulatory factor in the process of endometrial epithelial cell apoptosis in ruminants under inflammatory injury. The findings help elucidate the pathogenesis of postpartum endometritis in ruminants.

Main text

In ruminants, uterine infections occur frequently during the postpartum period. Endometritis is one of the principal causes of infertility in goats and dairy cows. The incidence of uterine infection in cattle and goats is 50% and 5.6%, respectively, and it causes substantial economic losses (Cui et al. 2020). Although the biology of the endometrium in different animals has unique mechanisms, pathogenic bacteria (such as Escherichia coli and Streptococcus) are the main cause of postpartum endometritis (Yin et al. 2021). Lymphocyte infiltration and increased macrophages and plasma cells in the lamina propria are common features of endometritis. Surface epithelial shedding and endometrial cell death are widely observed histopathological features (Beena et al. 2017). However, the precise mechanism linking endometritis to endometrial epithelial cell (EEC) apoptosis remains unclear.

Autophagy-related 7 (ATG7) is a core protein that regulates the classical autophagic degradation pathway through ATG8 lipidation. According to previous reports, autophagy-related functions of ATG7 participate in many physiological and pathological processes, such as the redistribution of tight junction proteins in EECs to preserve barrier function (Yang et al. 2022). Recent data have demonstrated that ATG7 regulates cell function independent of autophagy-associated signaling (Collier et al. 2021). Several studies have suggested that ATG7 is capable of regulating apoptosis, but surprisingly, ATG7 can both promote and inhibit apoptosis in different cellular contexts (He et al. 2021; Wang et al. 2021). To date, whether ATG7 mediates endometrial cell apoptosis in ruminants suffering from endometritis remains unclear.

In this study, we used an lipopolysaccharide (LPS) induced mouse endometritis injury model and a goat endometrial epithelial cell inflammation model to study ruminant endometritis. The location and expression level of ATG7 in mouse endometrial tissue under LPS treatment were analysed. To further explore the potential roles of ATG7 in EEC apoptosis, lentiviruses were selected to knock down or overexpress ATG7.

Increased expression of ATG7 in endometrial tissue of endometritis

To verify that the uteri suffered from inflammatory damage, histological analysis was performed using H&E staining. The abscission of endometrial luminal epithelial cells, necrosis and hemorrhages were observed in the diseased uterine tissues compared with healthy tissues (Fig. 1A). Fig. 1B showed that the fluorescence intensity of ATG7 in the endometrial luminal and glandular epithelial cells of the diseased uteri was greater than that in the healthy uteri. Western blot analysis also demonstrated that there was an increase in ATG7 expression in uterine tissues after LPS treatment (Fig. 1C). The above results provide evidence that ATG7 might participate in endometrial inflammatory injury.

Fig. 1

The expression of ATG7 in mouse uterine tissues. A H&E staining of uterine tissue. B The expression and location of ATG7 in uterine tissue. Red: ATG7. Blue: DAPI. C Western blot analysis of ATG7 protein levels with LPS treatment in mouse uterus tissues. LPS, lipopolysaccharide. The data are presented as the means ± SD of three independent experiments. * Significant difference (p < 0.05) compared with other groups. ** Significant difference (p < 0.01) compared with other groups. *** Significant difference (p < 0.001) compared with other groups

LPS induces high levels of ATG7 in goat EECs

Due to the increased expression of ATG7 in the diseased uterine tissues, we next wondered whether it could be upregulated in the endometrial inflammatory injury model in vitro. As shown in Fig. 2A, LPS increased ATG7 expression in goat EECs at 24 h. Similar results were obtained by Western blot: ATG7 expression was upregulated under LPS treatment in EECs at 12 and 24 h (Fig. 2B). Then, qPCR was used to examine the expression of apoptosis-related genes, including bcl-2 and bax, in EECs after treatment with LPS. Results showed that treatment reduced the mRNA levels of bcl-2 but increased bax expression in EECs at 24 h (Fig. 2C). Western blot analysis also confirmed these results (Fig. 2D). Similarly, there were more TUNEL-positive cells in the EECs treated with LPS than in those without LPS treatment, with a maximum at approximately 24 h after treatment (Fig. 2E). Therefore, the experimental conditions for LPS treatment were selected as 24 h to explore the roles of ATG7 in endometrial inflammatory injury.

Fig. 2

LPS-induced apoptosis in goat EECs. A and B The expression levels of ATG7 in LPS-treated goat EECs at different time points. C and D The expression levels of bcl-2 and bax in LPS-treated goat EECs at different time points. E Representative merged fluorescence images of TUNEL in goat EECs with or without LPS treatment. EECs, endometrial epithelial cell. LPS, lipopolysaccharide. The data are presented as the means ± SD of three independent experiments. * Significant difference (p < 0.05) compared with other groups. ** Significant difference (p < 0.01) compared with other groups. *** Significant difference (p < 0.001) compared with other groups

Silencing ATG7 inhibits apoptosis in goat EECs

Considering that LPS treatment increased ATG7 expression in EECs, whether ATG7 defects caused apoptosis were further explored. The interference efficiency of three constructed ATG7 lentivirus interference vectors were analyszed. As shown in Fig. 3A, the third ATG7 interfering vector (pCD513B-U6-ATG7-shRNA-3) significantly reduced the transcription level of ATG7. Western blotting further confirmed these results (Fig. 3B). Therefore, pCD513B-U6-ATG7-shRNA-3 were selected for subsequent testing. qPCR was performed to measure the levels of bax and bcl-2, the key genes responsible for regulating apoptosis. Silencing of ATG7 led to an increase in bcl-2 expression but declined bax expression in EECs (Fig. 3C and D). Correspondingly, silencing of ATG7 induced a reduction in apoptosis in EECs compared with shN under LPS treatment, as evidenced by decreased TUNEL staining (Fig. 3E).

Fig. 3

Silencing ATG7 inhibits apoptosis in goat EECs. A Real-time PCR analysis of atg7 mRNA levels in goat EECs. B Western blot analysis of ATG7 protein levels in goat. EECs, pCD513B-U6-ATG7-shRNA-1 and mock control were transduced for 48 h. C and D Real-time PCR and Western blot analysis of the bax, bcl-2 and ATG7 levels following LPS treatment. E Representative merged fluorescence images of TUNEL in goat EECs with or without LPS treatment. EECs, endometrial epithelial cell. LPS, lipopolysaccharide. The data are presented as the means ± SD of three independent experiments. * Significant difference (p < 0.05) compared with other groups. ** Significant difference (p < 0.01) compared with other groups. *** Significant difference (p < 0.001) compared with other groups

Overexpression ATG7 induces excessive apoptosis in goat EECs

To further validate the role of ATG7 in regulating the apoptosis of EECs with LPS treatment, ATG7 were overexpressed. ATG7-lentivirus infection, but not vector infection, produced a significant increase in ATG7 expression (Fig. 4A and B). ATG7 overexpression significantly increased the expression of bax and decreased bcl-2 expression (Fig. 4A and B). As shown in Fig. 4C, there were more TUNEL-positive cells in the ATG7-overexpressing EECs than in the vector EECs under LPS treatment. These results provide unequivocal evidence that ATG7 activation enhanced EEC apoptosis under LPS treatment.

Fig. 4

Overexpression ATG7 induces excessive apoptosis in goat EECs. A and B Real-time PCR and Western blot analysis of bax, bcl-2 and ATG7 following LPS treatment. C Representative merged fluorescence images of TUNEL in goat EECs with or without LPS treatment.EECs, endometrial epithelial cell. LPS, lipopolysaccharide. The data are presented as the means ± SDof three independent experiments. * Significant difference (p < 0.05) compared with other groups. ** Significant difference (p < 0.01) compared with other groups. *** Significant difference (p < 0.001) compared with other groups

Inflammatory injury of the endometrium is an obstetric disease with a high incidence in ruminants after delivery. The inflammatory uterus causes pain to female animals and is harmful to the next round of embryo implantation, which causes major economic losses to animal husbandry. Apoptosis of endometrial epithelial cells caused by excessive inflammation is a typical pathological feature of endometritis. Previous studies have found that ATG7 was involved in the process of apoptosis of various tissues and cells (She et al. 2022; Wang et al. 2022). Hence, in the present study, the relationship between ATG7 and apoptosis in EEC was investigated.

ATG7 is a key protein that regulates the function of goat endometrial epithelial cells (Yang et al. 2021). The loss of ATG7 led to inhibition of endometrial epithelial cell proliferation, decreased cell adhesion activity and decreased prostaglandin secretion (Yang et al. 2021). Yang reported that ATG7 plays a critical role in baicalin protecting the endometrial barrier by promoting the redistribution of tight junction proteins under LPS stimulation (Yang et al. 2022). The above studies suggest that ATG7 plays an important role in the physiological and pathological processes of the ruminant endometrium. However, whether ATG7 directly participates in the apoptosis of endometrial cells caused by endometritis injury has not been reported. In this study, we found for the first time that lipopolysaccharide-mediated endometrial inflammatory injury can lead to increased expression of ATG7 in endometrial luminal and glandular epithelial cells rather than endometrial stromal cells, suggesting that lipopolysaccharide might damage the integrity of the barrier of endometrial luminal epithelia and affect the secretion of uterine milk proteins.

Originally, autophagy was considered to be an important mechanism for protecting cells from apoptosis during nutritional deficiency. However, an increasing number of reports have found that sustained and excessive autophagy might also induce apoptosis (Wu et al. 2022). Liao demonstrated that there is crosstalk between apoptosis and autophagy (Liao et al. 2023). ATG7 is a well-known key autophagic protein that participates in classic autophagic degradation through ATG8 lipidation. Knocking out ATG7 caused an obstacle in the degradation of the inner autophagosomal membrane, which in turn blocked autophagic flux (Collier et al. 2021). As an essential link in autophagy, ATG7 undoubtedly plays an important role in regulating apoptosis. Previous studies have found that ATG7 knockout reduces cell viability and the proliferation rate in smooth muscle cells and confirmed that this effect is due to increased antioxidant gene expression and NRF2 nuclear translocation (Grootaert et al. 2015). Lee reported that ATG7 binds to p53 in a manner independent of its E1-like enzymatic activity, inhibiting the apoptosis of mouse embryonic fibroblasts and reducing the expression of proapoptotic genes (Bax, Puma and Noxa) (Lee et al. 2012). In contrast to earlier findings, the findings of the current study demonstrated that silencing ATG7 induced a reduction in apoptosis in goat EECs compared with shN under LPS treatment. In accordance with the present results, Karthikkeyan demonstrated that high levels of ATG7 inhibit the lysosomal-mediated degradation of autophagic vacuoles and promote apoptosis (Karthikkeyan et al. 2022). We speculate that ATG7-mediated EEC apoptosis may also be a similar mechanism under LPS treatment, but more studies need to be carried out to confirm this hypothesis.

In summary, this study showed that LPS induced increased ATG7 expression in endometrial epithelial cells rather than endometrial stromal cells. Knocking down ATG7 can reduce cell mortality and regulate the expression of apoptotic molecules in endometrial epithelial cells. Overexpression ATG7 further aggravated the apoptosis of goat endometrial epithelial cells induced by LPS. This study indicated that ATG7 may play a key regulatory role in the process of endometrial epithelial cell apoptosis in ruminants under inflammatory injury.

Methods

Experimental animal groups and treatments

Kunming female virgin mice of specific pathogen-free (SPF) grade at the age of approximately seven weeks were purchased from Hubei Provincial Center for Laboratory Animal Research, Wuhan, Hubei Province, China (approval number of the ethical proof of animal experiment: HZAUMO-2022-0058). The mice were housed in a temperature-controlled room (22 ± 2°C) with a 12 h light/12 h dark cycle and randomly acquired food and water. All experimental procedures were performed in strict accordance with the guidelines of the Animal Care and Use Committee of Huazhong Agricultural University. All experimental protocols involving animal subjects were approved by the Animal Care and Use Committee of Huazhong Agricultural University.

Mice (25–30 g) were randomly divided into two groups, with 10 mice in each group (n = 10), consisting of a control group and an LPS group. The method for constructing a mouse endometritis model was described previously (Liu, Wu et al. 2021). Twenty microliters of LPS (Sigma, St. Louis, MO, USA) at a concentration of 5 mg/mL was infused into the uterus to induce endometritis damage. Equal volumes of ddH₂O were administered into the uterus of the control group. At 24 h after LPS treatment, all mice were sacrificed (Fig. 1B).

Histological examination

The uteri were fixed with paraformaldehyde and then embedded in paraffin. The slides of uteri were dehydrated with gradient alcohol and subsequently stained with hematoxylin and eosin (H&E). The histological changes in the uteri were observed by an optical microscope (Olympus Shinjuku ku, Tokyo, Japan).

Immunofluorescence staining

The uteri were fixed with paraformaldehyde and then embedded in paraffin. The slides were dewaxed and rehydrated by subsequent immersion in xylene, ethanol (100%, 95%, 70% and 50%) and deionized H₂O. Antigen was then retrieved in citrate buffer. The slides of uteri were sequentially incubated with anti-ATG7 (ABclonal A0691, diluted 1:200) and Alexa-labeled secondary antibodies (Invitrogen, Life Technologies) at 37°C, and nuclei were counterstained with DAPI for 10 min. The localization and expression of ATG7 were observed in the uterus by a fluorescence microscope (Nikon, Inc., Melville, NY, USA).

Cell culture and cell transfection

Goat endometrial epithelial cells (EECs) are immortalized by transfection with human telomerase reverse transcriptase (Zhang, Wang et al. 2010). DMEM/F-12 medium containing 10% fetal bovine serum was used to culture EECs inoculated in a six-well plate. After treatment with LPS (2 μg/mL) for 6 h, 12 h or 24 h, EECs were collected for subsequent testing.

The ATG7 shRNA (shATG7), negative control (shN) and ATG7 overexpression vectors were constructed as previously reported (Yang et al. 2021; Yang et al. 2022). Supplementary Table S1 shows the sequences of shATG7 and shN. After lentivirus packaging, EECs were infected as previously reported (Chen, Lin et al. 2014).

RNA extraction and real-time quantitative PCR

After the total RNA was extracted, cDNA was synthesized according to the instructions (ABclonal Biotechnology, Wuhan, China). Supplementary Table 2 shows the primer sequences. Real-time quantitative PCR was performed using qPCR Mix (ABclonal Biotechnology, Wuhan, China) on a Roche LightCycler® 480 (Roche, USA). The GAPDH gene served as an internal control, and the expression levels of ATG7, bax and bcl-2 were estimated using the 2^−△△Ct method. Each real-time quantitative PCR experiment was performed with three independent samples.

Western blot analysis

Total protein was extracted using RIPA buffer. The EEC samples were separated by a 12% SDS‒PAGE gel, and the proteins were transferred to PVDF membranes (Millipore; Bedford, MA, USA). After incubation with 10% nonfat milk in Tris-buffered saline (TBS, pH 7.4)/0.1% Tween (TBST), the PVDF membranes were incubated with anti-ATG7 antibody (ABclonal A0691, diluted 1:1000), anti-BCL2 antibody (ABclonal A19693, diluted 1:1000), anti-BAX antibody (ABclonal A19684, diluted 1:1000) and anti-ACTB antibody (ABclonal AC038, diluted 1:2000) for 2 h at 37°C. Then, HRP-labeled secondary antibody was incubated with the PVDF membranes. Finally, the grayscale values of the protein bands were analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis

Unless otherwise specified, all data are expressed as the mean ± SD of three samples for each experimental point. All data were analyzed using SPSS V. 22 (IBM-SPSS, Inc., Chicago, USA). Significant differences between samples were analyzed using one-way ANOVA with the least significant difference (LSD), and the significance level was set as p < 0.05.