CTRP6 alleviates endometrial fibrosis by regulating Smad3 pathway in intrauterine adhesion†.

Intrauterine adhesion (IUA) is manifestations of endometrial fibrosis and excessive extracellular matrix deposition. C1q/tumor necrosis factor-related protein-6 (CTRP6) is a newly identified adiponectin paralog which has been reported to modulate the fibrosis process of several diseases; however, the endometrial fibrosis function of CTRP6 remains unknown. Our study aimed to assess the role of CTRP6 in endometrial fibrosis and further explore the underlying mechanism. Here, we found that the expression of CTRP6 was downregulated in the endometrial tissues of IUA. In vitro experiments demonstrated the reduced level of CTRP6 in facilitated transforming growth factor-ß1 (TGF-ß1)-induced human endometrial stromal cells (HESCs). In addition, CTRP6 inhibited the expression of α-smooth muscle actin (α-SMA) and collagen I in TGF-ß1-treated HESCs. Mechanistically, CTRP6 activated the AMP-activated protein kinase (AMPK) and protein kinase B (AKT) pathway in HESCs, and AMPK inhibitor (AraA) or PI3K inhibitor (LY294002) pretreatment abolished the protective effect of CTRP6 on TGF-ß1-induced fibrosis. CTRP6 markedly decreased TGF-ß1-induced Smad3 phosphorylation and nuclear translocation, and AMPK or AKT inhibition reversed these effects. Notably, CTRP6-overexpressing treatment alleviated the fibrosis of endometrium in vivo. Therefore, CTRP6 ameliorates endometrial fibrosis, among which AMPK and AKT are essential for the anti-fibrotic effect of CTRP6 via the Smad3 pathway. Taken together, CTRP6 may be a potential therapeutic target for the treatment of intrauterine adhesion.

Human amnion mesenchymal stem cells promote endometrial repair via paracrine, preferentially than transdifferentiation.

BACKGROUND: Intrauterine adhesion (IUA) is one of the most severe causes of infertility in women of childbearing age with injured endometrium secondary to uterine performance. Stem cell therapy is effective in treating damaged endometrium. The current reports mainly focus on the therapeutic effects of stem cells through paracrine or transdifferentiation, respectively. This study investigates whether paracrine or transdifferentiation occurs preferentially in treating IUA. METHODS: Human amniotic mesenchymal stem cells (hAMSCs) and transformed human endometrial stromal cells (THESCs) induced by transforming growth factor beta (TGF-ß1) were co-cultured in vitro. The mRNA and protein expression levels of Fibronectin (FN), Collagen I, Cytokeratin19 (CK19), E-cadherin (E-cad) and Vimentin were detected by Quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and Immunohistochemical staining (IHC). The Sprague-Dawley (SD) rats were used to establish the IUA model. hAMSCs, hAMSCs-conditional medium (hAMSCs-CM), and GFP-labeled hAMSCs were injected into intrauterine, respectively. The fibrotic area of the endometrium was evaluated by Masson staining. The number of endometrium glands was detected by hematoxylin and eosin (H&E). GFP-labeled hAMSCs were traced by immunofluorescence (IF). hAMSCs, combined with PPCNg (hAMSCs/PPCNg), were injected into the vagina, which was compared with intrauterine injection. RESULTS: qPCR and WB revealed that FN and Collagen I levels in IUA-THESCs decreased significantly after co-culturing with hAMSCs. Moreover, CK19, E-cad, and Vimentin expressions in hAMSCs showed no significant difference after co-culture for 2 days. 6 days after co-culture, CK19, E-cad and Vimentin expressions in hAMSCs were significantly changed. Histological assays showed increased endometrial glands and a remarkable decrease in the fibrotic area in the hAMSCs and hAMSCs-CM groups. However, these changes were not statistically different between the two groups. In vivo, fluorescence imaging revealed that GFP-hAMSCs were localized in the endometrial stroma and gradually underwent apoptosis. The effect of hAMSCs by vaginal injection was comparable to that by intrauterine injection assessed by H&E staining, MASSON staining and IHC. CONCLUSIONS: Our data demonstrated that hAMSCs promoted endometrial repair via paracrine, preferentially than transdifferentiation.

IUA is the crucial cause of infertility in women of childbearing age, and no satisfactory treatment measures have been found in the clinic. hAMSCs can effectively treat intrauterine adhesions through paracrine and transdifferentiation mechanisms. This study confirmed in vitro and in vivo that amniotic mesenchymal stem cells preferentially inhibited endometrial fibrosis and promoted epithelial repair through paracrine, thus effectively treating intrauterine adhesions. The level of fibrosis marker proteins in IUA-THESCs decreased significantly after co-culturing with hAMSCs for 2 days in vitro. However, the level of epithelial marker proteins in hAMSCs increased significantly, requiring at least 6 days of co-culture. hAMSCs-CM had the same efficacy as hAMSCs in inhibiting fibrosis and promoting endometrial repair in IUA rats, supporting the idea that hAMSCs promoted endometrial remodeling through paracrine in vivo. In addition, GFP-labeled hAMSCs continuously colonized the endometrial stroma instead of the epithelium and gradually underwent apoptosis. These findings prove that hAMSCs ameliorate endometrial fibrosis of IUA via paracrine, preferentially than transdifferentiation, providing the latest insights into the precision treatment of IUA with hAMSCs and a theoretical basis for promoting the "cell-free therapy" of MSCs.

Semaglutide May Ameliorate Fibrosis and Inhibit Epithelial-Mesenchymal Transition in Intrauterine Adhesion Models.

The purpose of this study was to explore the effect of Semaglutide on intrauterine adhesions and discover new drugs for such adhesions. In this study, the cell model was simulated by TGF-ß1-induced human endometrial epithelial cells, and the animal model was established through mechanical curettage and inflammatory stimulation. After co-culturing with TGF-ß1 with or without different concentrations of Semaglutide for 48 h, cells were collected for RT-qPCR and Western blotting analyses. Three doses were subcutaneously injected into experimental mice once a day for two weeks, while the control group received sterile ddH2O. The serum and uterine tissues of the mice were collected. HE and Masson staining were used for the uterine histomorphological and pathological analyses. RT-qPCR and Western blotting were used for mRNA and protein expression analyses. Serum indicators were detected using ELISA kits. The results showed that Semaglutide significantly reduced the mRNA levels of fibrosis indicators ACTA2, COL1A1, and FN and inflammatory indicators TNF-α, IL-6, and NF-κB in the two models. Semaglutide improved endometrium morphology, increased the number of endometrial glands, and reduced collagen deposition in IUA mice. The results also showed that Semaglutide could inhibit vimentin, E-Cadherin, and N-Cadherin in the two models. In summary, Semaglutide can ameliorate fibrosis and inflammation of intrauterine adhesions as well as inhibit epithelial-mesenchymal transition in IUA models.

Melatonin-Primed MSCs Alleviate Intrauterine Adhesions by Affecting MSC-Expressed Galectin-3 on Macrophage Polarization.

Intrauterine adhesion (IUA) is characterized by the presence of fibrosis in the uterine cavity. It is mainly caused by infection or trauma to the endometrium, and it imposes a great challenge to female reproductive health. Mesenchymal stem cells (MSCs) have been used to regenerate the human endometrium in patients with IUA, but stem cell therapy is not curative in some patients. Melatonin (MT) was reported as a potential modulator of MSCs. However, it remains unclear whether MSCs pretreated with MT exert an improved therapeutic effect on IUA. In this study, an IUA model was established using our invented electric scratching tool. Our results illustrated that MT-pretreated MSCs significantly attenuated the development of IUA. Moreover, MT-pretreated MSCs highly expressed galectin-3 (Gal-3), which enhanced MSC proliferation and migration and influenced macrophage polarization. Of note, IUA mice exhibited colonic injury, and MT-pretreated MSCs alleviated this injury by normalizing colonic microbial communities and recruiting macrophages. Furthermore, inhibition of sympathetic nerves had no effect on IUA progression but delayed colonic injury, and Gal-3 combined with norepinephrine better promoted M2-like macrophage polarization and inhibited M1-like macrophage polarization. Together, these data indicated that MT-primed MSCs can ameliorate injury of both the uterus and colon in an IUA model through high Gal-3 expression to influence sympathetic nerves and in turn affect the polarization and recruitment of macrophages.

Interleukin-34 accelerates intrauterine adhesions progress related to CX3CR1+ monocytes/macrophages.

Intrauterine adhesions (IUA) are characterized by endometrial fibrosis and impose a great challenge for female reproduction. IL-34 is profoundly involved in various fibrotic diseases through regulating the survival, proliferation, and differentiation of monocytes/macrophages. However, it remains unclear how IL-34 regulates monocytes/macrophages in context of IUA. Here, we showed that the expression level of IL-34 and the amount of CX3CR1+ monocytes/macrophages were significantly increased in endometrial tissues of IUA patients. IL-34 promoted the differentiation of monocytes/macrophages, which express CX3CR1 via CSF-1R/P13K/Akt pathway in vitro. Moreover, IL-34-induced CX3CR1+ monocytes/macrophages promoted the differentiation of endometrial stromal cells into myofibroblasts. Of note, IL-34 caused endometrial fibrosis and increased the amount of CX3CR1+ monocytes/macrophages in endometrial tissues in vivo. IL-34 modulated endometrial fibrosis by regulating monocytes/macrophages since the elimination of endometrial monocytes/macrophages significantly suppressed the profibrotic function of IL-34. Finally, blocking of IL-34 in the LPS-IUA model resulted in the improvement of endometrial fibrosis and decreased number of CX3CR1+ monocytes/macrophages. Our studies uncover the novel mechanism of interaction between IL-34-induced CX3CR1+ monocytes/macrophages and endometrial stromal cells in endometrial fibrosis pathogenesis, and highlight IL-34 as a critical target for treating IUA.

Development of a refined ex vivo model of peritoneal adhesion formation, and a role for connexin 43 in their development.

Despite many advances across the surgical sciences, post-surgical peritoneal adhesions still pose a considerable risk in modern-day procedures and are highly undesirable. We have developed a novel mouse peritoneal strip ex vivo adhesion model which may serve to bridge the gap between single cell culture systems and in vivo animal drug testing for the assessment of potential anti-adhesion agents, and study of causality of the process. We investigated the optimal conditions for adhesion formation with mouse peritoneal tissue strips by modifying an existing ex vivo rat model of peritoneal adhesions. We assessed the impact of the following conditions on the formation of adhesions: contact pressure, abrasions, and the presence of clotted blood. Macroscopic adhesions were detected in all mouse peritoneal strips exposed to specific conditions, namely abrasions and clotted blood, where peritoneal surfaces were kept in contact with pressure using cotton gauze in a tissue cassette. Adhesions were confirmed microscopically. Interestingly, connexin 43, a gap junction protein, was found to be upregulated at sites of adhesions. Key features of this model were the use of padding the abraded tissue with gauze and the use of a standardised volume of clotted blood. Using this model, peritoneal strips cultured with clotted blood between abraded surfaces were found to reproducibly develop adhesion bands at 72 h. Our goal is to develop a model that can be used in genetically modified mice in order to dissect out the role of particular genes in adhesion formation and to test drugs to prevent adhesion formation.

E8002 Reduces Adhesion Formation and Improves Joint Mobility in a Rat Model of Knee Arthrofibrosis.

Knee arthrofibrosis is a common complication of knee surgery, caused by excessive scar tissue, which results in functional disability. However, no curative treatment has been established. E8002 is an anti-adhesion material that contains L-ascorbic acid, an antioxidant. We aimed to evaluate the efficacy of E8002 for the prevention of knee arthrofibrosis in a rat model, comprising injury to the surface of the femur and quadriceps muscle 1 cm proximal to the patella. Sixteen male, 8-week-old Sprague Dawley rats were studied: in the Adhesion group, haemorrhagic injury was induced to the quadriceps and bone, and in the E8002 group, an adhesion-preventing film was implanted between the quadriceps and femur after injury. Six weeks following injury, the restriction of knee flexion owing to fibrotic scarring had not worsened in the E8002 group but had worsened in the Adhesion group. The area of fibrotic scarring was smaller in the E8002 group than in the Adhesion group (p < 0.05). In addition, the numbers of fibroblasts (p < 0.05) and myofibroblasts (p < 0.01) in the fibrotic scar were lower in the E8002 group. Thus, E8002 reduces myofibroblast proliferation and fibrotic scar formation and improves the range of motion of the joint in a model of knee injury.

Management of intrauterine adhesions using human amniotic mesenchymal stromal cells to promote endometrial regeneration and repair through Notch signalling.

Intrauterine adhesions (IUAs) severely hamper women's reproductive functions. Human amniotic mesenchymal stromal cell (hAMSC) transplantation is effective in treating IUAs. Here, we examined the function of Notch signalling in IUA treatment with hAMSC transplantation. Forty-five Sprague-Dawley female rats were randomly divided into the sham operation, IUA, IUA + E2, IUA + hAMSCs and IUA + hAMSCs + E2 groups. After IUA induction in the rats, hAMSCs promoted endometrial regeneration and repair via differentiation into endometrial epithelial cells. In all groups, the expression of key proteins in Notch signalling was detected in the uterus by immunohistochemistry. The results indicated Notch signalling activation in the hAMSCs and hAMSCs + E2 groups. We could also induce hAMSC differentiation to generate endometrial epithelial cells in vitro. Furthermore, the inhibition of Notch signalling using the AdR-dnNotch1 vector suppressed hAMSC differentiation (assessed by epithelial and mesenchymal marker levels), whereas its activation using the AdR-Jagged1 vector increased differentiation. The above findings indicate Notch signalling mediates the differentiation of hAMSCs into endometrial epithelial cells, thus promoting endometrial regeneration and repair; Notch signalling could have an important function in IUA treatment.

Antithrombin Together with NETs Inhibitor Protected Against Postoperative Adhesion Formation in Mice.

BACKGROUND/AIMS: Postoperative adhesions may induce adverse outcomes in patients. Adhesion formation is initiated by fibrin accumulation at the surgical site which is followed by local neutrophilia and the establishment of neutrophil extracellular traps (NET). Previous reports have suggested that the preventive efficacy of reagents designed to reduce postoperative adhesion is inversely correlated with neutrophilia and NET production. Antithrombin (AT) is a natural inhibitor of thrombin, a key factor in coagulation. Here, we evaluate whether treatment with AT and/or NET inhibitors prevent or reduce postoperative adhesion formation in mice. METHODS: Mice were treated with AT and/or NET inhibitors before and/or after cecum cauterization and their adhesion scores were evaluated on day 7 post-operation. Immunochemistry/ immunofluorescence analyses were also performed and we used GSK484, an inhibitor of peptidyl arginine deiminase 4 (PAD4), as the NET inhibitor. RESULTS: AT or GSK484 partially rescued postoperative adhesion formation in mice. AT prevented thrombin-induced plasminogen activator inhibitor 1 and interleukin-6 expression in mesothelial cells in vitro. However, AT could not prevent neutrophilia or NETs formation around the injured serosa. Finally, we investigated a combination of AT and a PAD4 inhibitor and found that this could inhibit almost all adhesion formation in these animals. Since AT-inactivating proteases are liberated following NET release, they might dampen the biological action of the AT treatment. This suggests that NET inhibitors might allow AT to exert its full action in the surgically injured serosa. CONCLUSION: Combined treatment with AT and GSK484 may effectively attenuate postoperative adhesion production in mice.

An in vitro system to investigate IOL: Lens capsule interaction.

Posterior capsule opacification (PCO) is the most common complication associated with intraocular lens (IOL) implantation. Unfortunately, current in vitro models cannot be used to assess the potential of PCO due to their failure to simulate the posterior curvature of the lens capsule (LC) and IOL, a factor known to affect PCO pathogenesis in clinic. To overcome such a challenge, a new system to study IOL: LC interaction and potentially predict PCO was developed in this effort. It is believed that the interactions between an IOL and the lens capsule may influence the extent of PCO formation. Specifically, strong adhesion force between an IOL and the LC may impede lens epithelial cell migration and proliferation and thus reduce PCO formation. To assess the adhesion force between an IOL and LC, a new in vitro model was established with simulated LC and a custom-designed micro-force tester. A method to fabricate simulated LCs was developed by imprinting IOLs onto molten gelatin to create simulated three dimensional (3D) LCs with curvature resembling the bag-like structure that collapses on the IOL post implantation. By pushing the LC mold vertically downward, while measuring the change in position of the bending bar with respect to its start position, the adhesion force between the IOLs and LCs was measured. An in vitro system that can measure the adhesion force reproducibly between an IOL and LC with a resolution of ~1 µN was established in this study. During system optimization, the 10% high molecular weight gelatin produced the best LC with the highest IOL: LC adhesion force with all test lenses that were fabricated from acrylic foldable, polymethylmethacrylate (PMMA) and silicone materials. Test IOLs exerted different adhesion force with the 3D simulated LCs in the following sequence: acrylic foldable IOL > silicone IOL > PMMA IOL. These results are in good agreement with the clinical observations associated with PCO performance of IOLs made of the same materials. This novel in vitro system can provide valuable insight on the IOL: LC interplay and its relationship to clinical PCO outcomes.

Effect of time and temperature-dependent changes of IOL material properties on IOL: Lens capsule interactions.

Posterior Capsule Opacification (PCO) is the most common complication associated with Intraocular Lens (IOL) implantation. Based on the assumption that the interactions between an IOL and the lens capsule (LC) may influence the extent of PCO formation, a new in vitro model was developed to quantify the adhesion force of an IOL to simulated LC using a custom-designed micro-force tester. Using this system, we examined the influence of temperature (room temperature vs. body temperature) and incubation time (0 vs. 24 h) on the adhesion force between IOLs and LCs. The results show that, in line with clinical observations of PCO incidence, the adhesion force increased at body temperature and with increase in incubation time in the following order, Acrylic foldable IOLs > Silicone IOLs > PMMA IOLs. By examining the changes of surface properties as a function of temperature and incubation time, we found that acrylic foldable IOLs showed the largest increase in their hydrophilicity and reported the lowest surface roughness in comparison to other IOL groups. Coincidentally, using a newly established macromolecular dye imaging system to simulate cell migration between IOLs and LC, we observed that the amount of macromolecular dye infiltration between IOLs and LCs was in the following order: PMMA IOLs > Silicone IOLs > Acrylic foldable IOLs. These results support a new potential mechanism that body temperature, incubation time, surface hydrophilicity and smoothness of IOLs greatly contribute to their tight binding to LCs and such tight binding may lead to reduced IOL: LC space, cell infiltration, and thus PCO formation.

The anti-adhesive effect of anti-VEGF agents in experimental models: A systematic review.

Adhesions constitute a major problem in abdominal-pelvic and thoracic surgery with significant impact in the postoperative quality of life and healthcare services utilization. Adhesiogenesis is the result of increased fibrin formation, impaired fibrinolysis, angiogenesis, and fibrosis. Despite the recent advancements, the ideal anti-adhesive agent remains to be determined. To this end, we performed a comprehensive literature search in PubMed, EMBASE, and Scopus databases to identify studies investigating the antiadhesive role of anti-VEGF agents in peritoneal, pleural, and pericardial experimental adhesion models. Fifteen studies were eligible for inclusion with a total population of 602 animals (334 rats, 180 rabbits, and 88 mice). The majority of included studies (11/15) used bevacizumab, while three studies used other anti-VEGF antibodies and one study used an anti-VEGFR-antibody. A rat model was used in nine studies, while rabbit (n = 3) or mouse (n = 3) models were used less frequently. Eleven studies used peritoneal models, three studies used pleural models, and one study used a pericardial model. The scales (n = 12) and interval (Range: 1-42 days) used for the evaluation of adhesions varied between the studies. All studies demonstrated a significant decrease in adhesion scores between the anti-VEGF and control groups up to 42 days postprocedure. VEGF blockade resulted in decreased fibrosis in four out of five studies that used peritoneal models, while the effect on pleural models depended on the pleurodesis agent and was significant between 7 and 28 days. The effect of anti-VEGF agents on anastomosis integrity depends on the dose and the model that is used (inconclusive results).Current data support the anti-adhesive role of Anti-VEGF agents in all three serosal surfaces up to 6 weeks postprocedure. Further studies are needed to confirm the anti-adhesive role of anti-VEGF agents in pleural and pericardial adhesion experimental models and investigate any effect on anastomosis integrity in peritoneal models.

REVISED PREDICTIVE BIOMARKER FOR OCRIPLASMIN THERAPY IN VITREOMACULAR TRACTION DISORDERS.

PURPOSE: To determine a statistically optimal limit of adhesion size in vitreomacular traction (VMT) syndrome for ocriplasmin treatment. METHODS: In this retrospective, consecutive, interventional study, we included 106 patients treated with ocriplasmin injection because of VMT between July 2013 and January 2018. A univariate and multivariate risk analysis was performed with grouped factors and continuous factors. We used a receiver operating characteristic curve to measure the prognostic relevance of each continuous factor for therapy success and determined the statistically optimal cutoff value at which specificity and sensitivity are simultaneously maximized. RESULTS: Among the grouped factors, only a phakic lens status showed a highly significant positive influence on the resolution of the VMT. For the continuous factors, only the adhesion diameter before injection was a good predictor of anatomical success. The statistically optimal threshold value for the adhesion size was calculated to be 480 µm. Eyes below this limit had a 6.84-fold better chance of VMT resolution compared with eyes with a larger adhesion diameter. CONCLUSION: The threshold value of the VMT diameter for ocriplasmin therapy could be statistically defined as 480 µm and may thus be a new quantitative biomarker to predict treatment success.

Human Acellular Amniotic Matrix with Previously Seeded Umbilical Cord Mesenchymal Stem Cells Restores Endometrial Function in a Rat Model of Injury.

BACKGROUND: Abnormal endometrial repair after injury results in the formation of intrauterine adhesions (IUA) and a thin endometrium, which are key causes for implantation failure and infertility. Stem cell transplantation offers a potential alternative for some cases of severe Asherman's syndrome that cannot be treated with surgery or hormonal therapy. Umbilical cord-derived mesenchymal stem cells (UCMSCs) have been reported to repair the damaged endometrium. However, there is no report on the effects of UCMSCs previously seeded on human acellular amniotic matrix (AAM) on endometrial injury. METHODS: Absolute ethanol was injected into rat uteri to damage the endometrium. UCMSCs previously seeded on AAM were surgically transplanted. Using a variety of methods, the treatment response was assessed by endometrial thickness, endometrial biomarker expression, endometrial receptivity, cell proliferation, and inflammatory factors. RESULTS: Endometrial thickness was markedly improved after UCMSC-AAM transplantation. The expression of endometrial biomarkers, namely, vimentin, cytokeratin, and integrin ß3, in treated rats increased compared with untreated rats. In the UCMSC-AAM group, the VEGF expression decreased, whereas that of MMP9 increased compared with the injury group. Moreover, in the AAM group, the MMP9 expression increased. The expression of proinflammatory factors (IL-2, TNFα, and IFN-γ) in the UCMSC-AAM group decreased compared with the untreated group, whereas the expression of anti-inflammatory factors (IL-4, IL-10) increased significantly. CONCLUSIONS: UCMSC transplantation using AAM as the carrier can be applied to treat endometrial injury in rats. The successful preparation of lyophilized AAM provides the possibility of secondary infectious disease screening and amniotic matrix quality detection, followed by retrospective analysis. The UCMSC-AAM complex may promote the better application of UCMSCs on the treatment of injured endometrium.

Standardised pomegranate peel extract lavage prevents postoperative peritoneal adhesion by regulating TGF-ß and VEGF levels.

Peritoneal adhesion represents a severe complication following surgery. Punica granatum (pomegranate) possesses several anti-oxidative and anti-inflammatory properties. Pomegranate peel extract (PPEx) can alleviate the production of various inflammatory factors and cytokines. Thus, we sought to evaluate the anti-adhesion effects of pomegranate in rats. Thirty male Wistar rats (6-week-old, 220 ± 20 g) were divided into five groups (n = 6): normal group without any surgical procedures, control group, and experimental groups receiving 2 ml of 1%, 2%, and 4% w/v PPEx, respectively. Peritoneal adhesions were examined macroscopically. Furthermore, we evaluated inflammatory cytokines levels [interleukin 6 (IL-6), and tumour necrosis factor-α (TNF-α)], growth factors [transforming growth factor- ß1 (TGF-ß1), and vascular endothelial growth factor (VEGF)], and oxidative stress parameters [nitric oxide metabolites (NO), and malondialdehyde (MDA), and glutathione (GSH)] using biochemical methods. Our results showed that the adhesion score and IL-6, TNF-α, TGF-ß1, VEGF, NO, and MDA levels were increased in the control group. In contrast, the GSH level was diminished in the control group compared with the normal group (P < 0.001). PPEx (1 and 2% w/v) markedly reduced all measured parameters compared with the control group (P < 0.001-0.05). PPEx may reduce peritoneal adhesion by alleviating adhesion formation, IL-6, TNF-α, TGF-ß1, VEGF, NO, and MDA, and stimulating anti-oxidative factors. Therefore, PPEx may be considered an appropriate candidate for the treatment of postoperative peritoneal adhesion.

Functional analysis of miRNAs combined with TGF-ß1/Smad3 inhibitor in an intrauterine rat adhesion cell model.

In this study, we aimed to study the role of miRNAs in intrauterine adhesion (IUA) disease. An IUA cell model was constructed by TGF-ß1. Smad3 inhibitor (SIS3) can inhibit the Smad3 signaling pathway and affect the role of TGF-ß1; thus, it was used to identify the role of Smad3 and related miRNAs in IUA. Cell number significantly increased in the TGF-ß1 group after 72 h and 96 h, respectively, compared with that in the control group (P < 0.05). However, cell proliferation was significantly decreased in the TGF-ß1 + SIS3 group (P < 0.0001). Cell apoptosis was increased in the TGF-ß1 + SIS3 group compared with that in the TGF-ß1 group. Western Blot (WB) analysis suggested that TGF-ß1 treatment could effectively increase the expression of α-SMA, COL1, Smad3, and p-Smad3, which could be inhibited by SIS3 treatment. A total of 235 and 530 differentially expressed miRNAs in the TGF-ß1 + SIS3 group were significantly up- and downregulated compared with those in the TGF-ß1 group, respectively. These differentially expressed miRNAs were enriched in the MAPK and PI3K-AKT pathways. The ten most differentially expressed miRNAs were selected to verify their expressions using quantitative real-time polymerase chain reaction (qPCR). Furthermore, overexpression of rno-miR-3586-3p and rno-miR-455-5p can promote cell proliferation and exacerbate the IUA pathogenic process. However, overexpression of rno-miR-204-3p and rno-miR-3578 can inhibit cell behavior and IUA progression. The above results can provide detailed information for the understanding of IUA molecular mechanisms.

CircPlekha7 plays an anti-fibrotic role in intrauterine adhesions by modulating endometrial stromal cell proliferation and apoptosis.

Circular RNA (circRNA) plays a key role in the development and progression of several diseases; however, its role in intrauterine adhesions (IUAs) is not well understood. This study aims to investigate the expression profiles and potential role of circRNA in IUA. RNA-sequencing was performed to screen for abnormally expressed circRNAs in TGF-ß1-induced IUA endometrial stromal cell (ESC) model (IUA group) and an SMAD3 inhibitor, SIS3-treated IUA ESC model (SIS3 group). Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to uncover the key functions and pathways. Interaction networks were constructed and analyzed based on the competing endogenous RNA hypothesis of circRNA. CircRNAs were validated by Sanger sequencing and quantitative polymerase chain reaction (qPCR). Cell proliferation and apoptosis were measured using MTS and flow cytometry, respectively. The protein and mRNA expression levels of fibrosis-related proteins were measured using western blotting and reverse transcription-qPCR, respectively. A total of 66 circRNAs were differentially expressed between the IUA and SIS3 groups. CircPlekha7 was identified as one of the significantly upregulated circRNAs in the SIS3 group. Overexpression of circPlekha7 enhanced apoptosis, decreased the viability of ESCs, and suppressed the expression of α-SMA, collagen I, and SMAD3 in ESCs; whereas knockdown of circPlekha7 exhibited opposite results. Altogether, the results indicate that circPlekha7 plays an anti-fibrotic role in IUA and may serve as a promising prognostic biomarker for patients with IUA. Therefore, overexpression of circPlekha7 could be a potential treatment strategy for IUA.

LncRNA HOTAIR promotes endometrial fibrosis by activating TGF-ß1/Smad pathway.

Homeobox transcript antisense RNA (HOTAIR) is a long non-coding RNA associated with a number of fibrosis-related diseases. The aim of this study was to investigate the specific role of HOTAIR in the development of endometrial fibrosis and to identify the molecular mechanisms underlying this process. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression levels of HOTAIR in samples of intrauterine adhesion (IUA) tissue and in endometrial stromal cells (ESCs) that had been treated with transforming growth factor beta 1 (TGF-ß1). Additionally, we transfected ESCs with either overexpression plasmid (pcDNA-HOTAIR) or silencing construct (si-HOTAIR) and then treated these cells with TGF-ß1. We then performed RT-qPCR and western blot analysis, along with cell proliferation and apoptosis assays, to investigate the effects of HOTAIR on the transdifferentiation of ESCs into myofibroblasts. The results showed that the expression levels of HOTAIR were significantly elevated in IUA tissue and in ESCs that had been treated with TGF-ß1. The overexpression of HOTAIR had a pro-fibrotic effect on ESCs, while the silencing of HOTAIR exerted an anti-fibrotic effect. Most importantly, the protein expression levels of p-Smad2 and p-Smad3 were significantly upregulated in TGF-ß1-treated ESCs transfected with pcDNA-HOTAIR and were downregulated after transfection with si-HOTAIR constructs. These data indicate that HOTAIR promotes endometrial fibrosis by activating the TGF-ß1/Smad signaling pathway, suggesting that the inhibition of HOTAIR may represent a promising therapeutic option for suppressing endometrial fibrosis.

20(S)-Ginsenoside Rg3-loaded electrospun membranes to prevent postoperative peritoneal adhesion.

Postoperative peritoneal adhesions are one of the most common surgical complications. In this study, we developed a 20(S)-ginsenoside Rg3-loaded methoxy poly (ethylene glycol)-block-poly(L-lactide-co-glycolide) (mPEG-b-PLGA) electrospun membrane (PEM/Rg3) that could not only serve as a physical barrier, but also as a drug delivery system that releases 20(S)-ginsenoside Rg3 constantly to prevent postoperative peritoneal adhesions. The characteristics of PEM/Rg3, including scanning electron microscopy, water contact angle, and mechanical analyses, were assessed. Degradation and drug release assays of PEM/Rg3 were performed. The anti-adhesion efficacy of PEM/Rg3 was evaluated in an abdomen-cecum mouse model. The adhesion scores, adhesion areas, hematoxylin and eosin (H&E) staining, immunofluorescence, and western blotting were assessed. The 20(S)-ginsenoside Rg3 loaded mPEG-b-PLGA electrospun fibers were successfully fabricated. The fibers were smooth, with no obvious drug crystals. PEM/Rg3 membranes were biodegradable and could be degraded gradually to release 20(S)-Ginsenoside Rg3 constantly from the membranes. The animal study showed that PEM/Rg3 exhibited an excellent adhesion prevention ability when compared with the control group, the PEM group, and polylactic acid (PLA) commercial membrane (Surgiwrap™) group. Immunofluorescence and western blotting studies showed that PEM/Rg3 inhibited the expressions of interleukin 1 (IL-1), interleukin 6 (IL-6), and reactive oxygen species modulator-1 (ROMO1). The 20(S)-ginsenoside Rg3-loaded mPEG-b-PLGA electrospun membranes exhibited satisfactory anti-adhesion efficacy by inhibiting inflammatory responses and oxidative stress. This composite represents a promising strategy to prevent postoperative peritoneal adhesions.

Developmental endothelial locus-1 prevents development of peritoneal adhesions in mice.

Postoperative peritoneal adhesions, fibrous bands formed in the peritoneal cavity following surgery, represent a common, challenging and costly problem faced by surgeons and patients, for which effective therapeutic options are lacking. Since aberrant inflammation is one of the key mechanisms underlying peritoneal adhesion formation, here we set out to study the role of developmental endothelial locus-1 (Del-1), which has been recently identified as an endogenous inhibitor of inflammation, in the formation of postoperative peritoneal adhesions using a mouse model of peritoneal adhesions induced by ischemic buttons. Del-1-deficient mice had a higher incidence of adhesions, and their adhesions had higher quality and tenacity scores. Del-1 deficiency also led to enhanced inflammation mediators and collagen production. Finally, Del-1 supplementation decreased the incidence and severity of postoperative peritoneal adhesions. Taken together, these results indicate a protective role for Del-1 in postoperative peritoneal adhesion formation.