Improvement in post-partum uterine involution in rats treated with Apios americana

Improvement in post-partum uterine involution in rats treated with Apios americana 美国豆芋加快产后大鼠的子宫修复 1 first-author 5 1 1 1 2 3 4 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xdzheng@zju.edu.cn †E-mail:xdzheng@zju.edu.cn xdzheng@zju.edu.cn College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China National Engineering Laboratory of Intelligent Food Technology and Equipment, Hangzhou 310058, China National Engineering Laboratory of Intelligent Food Technology and Equipment, Hangzhou 310058, China Zhejiang Key Laboratory for Agro-Food Processing, Hangzhou 310058, China Zhejiang Key Laboratory for Agro-Food Processing, Hangzhou 310058, China Fuli Institute of Food Science, Hangzhou 310058, China Fuli Institute of Food Science, Hangzhou 310058, China Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310022, China Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310022, China Objective: 2 Apios americana , a plant used as a staple ingredient of native American diets, has various properties, including anti-cancer, anti-hyperglycemic, hypotensive, and anti-inflammatory activity. In Japan, Apios is used as a post-natal medication. After parturition, women undergo a period of recovery as they return to pre-pregnancy conditions. However, few health products that aid post-partum recovery are on the market. We explored whether Apios can accelerate the post-partum recovery process, in particular the involution of the uterus. Methods: 2 Female rats kept in individual cages were mated with two male rats, with the exception of the control group (female rats without mating, on basal diet; n =6). After delivery, rats were divided into five groups based on their diet: basal diet (model; n =6); basal diet+oral intake at 5.4 g/kg of Chanfukang granules (a Chinese patent medicine preparation for post-partum lochia) (positive; n =6); basal diet containing 10% Apios powder (low; n =6); basal diet containing 20% Apios powder (medium; n =6); basal diet containing 40% Apios powder (high; n =6). Five days later, uteri and spleens were weighed. Uterus and spleen indices for each rat were calculated by dividing visceral weight by the total weight. Hormone and cytokine concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Histological analysis of uteri was completed using hematoxylin and eosin (H & E) staining. Expression of matrix metalloproteinases and inhibitors in uteri was measured by western blotting. Results: 2 Our results showed that Apios treatment reduced the post-partum uterus index and regulated the hormone concentrations. Moreover, we found that the process of uterine involution was accelerated, based on morphological changes in the uterus. In addition, our results indicated that Apios alleviated the inflammatory response induced by the involution process. Transforming growth factor β was also found to be regulated by Apios. There were significant downregulation of matrix metalloproteinases and upregulation of their inhibitors by Apios, which suggested that Apios increased the rate of the collagen clearance process. Conclusions: 2 These results, based on experimental observations at the molecular and protein levels, verified our hypothesis that Apios can improve uterine involution, and demonstrated the potential application of Apios in post-partum care. 目的 2 研究美国豆芋( Apios americana )对产后恢复过程,特别是子宫复旧的影响. 创新点 2 开发针对产后恢复的新食品. 方法 2 将雌雄大鼠配种交配,对照组除外(没有交配的雌性大鼠,基础日粮, n =6).分娩后,将雌性大鼠分为五组:基础饮食(模型组, n =6);基础饮食+灌胃给药5.4 g/kg的产妇康颗粒(阳性组, n =6);含10%豆芋粉末的基础日粮(低剂量组, n =6);含20%豆芋粉末的基础日粮(中剂量组, n =6);含40%豆芋粉末的基础日粮(高剂量组, n =6).实验结束后,称量子宫和脾脏质量,计算子宫和脾脏指数.并测量血清内激素和细胞因子水平,研究子宫的组织病理学变化,检测基质金属蛋白酶和抑制剂在子宫中的表达. 结论 2 豆芋干预减少了产后子宫指数并调节了激素浓度,加速了子宫形态复旧的过程,减轻了分娩引起的炎症反应.同时发现,转化生长因子β也受豆芋调节.豆芋干预使得基质金属蛋白酶显著下调,其抑制剂则显著上调,这表明豆芋增加了胶原清除过程的速度. Apios americana Uterine involution Cytokine Matrix metalloproteinase 美国豆芋 子宫复旧 细胞因子 基质金属蛋白酶 Introduction 1 The uterus undergoes dramatic changes throughout adult life, particularly during pregnancy. After delivery, the uterus begins remodeling to return to pre-pregnancy size and condition through the process of “uterine involution.” However, impaired tissue processes may lead to gynecological complications, such as post-partum hemorrhage, infections, and placental abnormalities (Rosen, 2008 2008 ; WHO, 2018 2018 ). About 830 women die from preventable diseases related to pregnancy and childbirth globally every day. In 2015, roughly 303 000 women died during pregnancy and following childbirth (Alkema et al., 2016 2016 ). There is an urgent, unmet clinical need to reduce the incidence of maternal mortality and uterine sub-involution. Post-partum care is important for accelerating the process of involution and avoiding the appearance of post-partum complications, for instance, injecting oxytocin for promoting uterine contractions to avoid the risk of bleeding (Wang et al., 2017 2017 ; WHO, 2018 2018 ), or giving an adequate dose of iron to avoid anemia (WHO, 2015 2015 ). However, few functional foods are available to aid post-partum uterine involution, although pineapple has been suggested as having a positive impact (Harianja et al., 2017 2017 ). Apios americana , a perennial vine with edible beans and tubers (Reynolds et al., 1988 1988 ), was a plant used as a staple ingredient of native American diets. Now it is widely used in many countries, especially in Japan (Hoshikawa and Juliarni, 1995 1995 ). It contains a high level of protein (Morales and Foster, 2009 2009 ), good-quality starch (Kikuta et al., 2012 2012 ), and a wide variety of fatty acids and minerals (Wilson et al., 1986 1986 ; Kinugasa and Watanbe, 1992 1992 ). In addition to the main nutrients, secondary metabolites, including isoflavones and saponins, are also found at high levels in Apios (Krishnan, 1998 1998 ; Nara et al., 2011 2011 ; Chitisankul et al., 2015 2015 ). It is reported that Apios and its extracts have antioxidant activity (Takashima et al., 2013 2013 ) and anti-cancer (Zhang et al., 2011 2011 ), anti-hyperglycemic (Kawamura et al., 2015 2015 ; Yan et al., 2017 2017 ), hypotensive (Kuramoto et al., 2013 2013 ), and anti-inflammatory effects (Cui et al., 2016 2016 ). Apios can also be used as a post-natal medication and for fatigue relief (Hoshikawa and Juliarni, 1995 1995 ). This study aimed to determine the influence and mechanism of action of Apios on the process of uterine involution. During uterine involution, extracellular matrix (ECM) remodeling is required, including cell proliferation and apoptosis (Takamoto et al., 1998 1998 ; Shkurupiy et al., 2011 2011 ). Most common mediators known to be involved in uterine involution appear to be similar to those involved in menstruation (Salamonsen, 2003 2003 ), including cytokines (Galvão et al., 2011 2011 ), growth factors (Yoshii et al., 2014 2014 ), and matrix-degrading enzymes (Takamoto et al., 1998 1998 ; Nguyen et al., 2016 2016 ). We hypothesized that Apios accelerates uterine involution. Therefore, in this study, we investigated endometrial and myometrial changes via direct measurements of uterine weight and hematoxylin and eosin (H & E) staining of post-partum rat uteri. We also investigated endocrine changes by determining the hormones, growth factors, and cytokines related to inflammatory responses in serum. Finally, we explored the activity of matrix metalloproteinases (MMPs) and the tissue inhibitor of metalloproteinase 1 (TIMP-1) at the protein level in uteri by western blotting. Materials and methods 1 Sample preparation 2 Tubers of Apios farmed in Hangzhou, Zhejiang Province, China were used for this study. A ground powder was obtained from boiled tubers after hot air drying at 50 °C. Animals 2 The experimental protocols were approved by the Ethics Committee of Laboratory Animal Care and Welfare, School of Medicine, Zhejiang University, Hangzhou, China. All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Female Sprague-Dawley rats weighing 200–300 g (8–10 weeks old) were obtained from the Experimental Animal Centre of Zhejiang Academy of Medicine Sciences, Hangzhou, China, certificate No. SCXK(Zhe)2017-0001. The animals were housed in a breeding room at a controlled temperature (20–24 °C) and with a 12-h light/dark cycle. Animals were allowed free access to basal food and water before beginning the experiment. The female rats, kept in individual cages, were mated with two male rats with the exception of the control group (female rats without mating, on basal diet; n =6). The formation of a vaginal plug indicated the first day of pregnancy. After delivery, rats were divided into five groups based on their diet: basal diet (model; n =6); basal diet+oral intake at 5.4 g/kg of Chanfukang granules (a Chinese patent medicine preparation for post-partum lochia) (positive; n =6); basal diet containing 10% Apios powder (low; n =6); basal diet containing 20% Apios powder (medium; n =6); basal diet containing 40% Apios powder (high; n =6). Tissue collection 2 Rats in the six groups were given free access to the above experimental diets for five days. During the experiment, body weight and dietary intake were measured. After five days of dietary intervention, the rats were fasted for 12 h and then anaesthetized after intra-peritoneal injection of 10% chloral hydrate (0.3 mL/100 g), and then blood samples from the abdominal artery were collected into tubes and centrifuged to separate the serum. Uteri and spleens were also promptly removed and weighed. Uterus and spleen indices were calculated as the visceral weight measurements divided by the total weight of each animal. Enzyme-linked immunosorbent assays (ELISA) 2 Serum concentrations of estrogen (E), progesterone (P), tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), IL-6, immunoglobulin A (IgA), IgG, transforming growth factor β1 (TGF-β1), and TGF-β3 were analyzed using commercially available assay kits (Elabscience Biotechnology, Wuhan, China). Histological analysis 2 Half of the “Y” type uteri were immediately fixed in 10% buffered paraformaldehyde for 24 h at 4 °C. Subsequently, standard pieces of uteri were embedded in paraffin after sequential dehydration in a graded ethanol series, and sliced into 5-μm thick sections before being stained with H&E stain. After H&E staining, the thickness of the endometrium was measured using a light microscope with a calibrated ocular scale. Western blot analysis 2 Total uterine protein extracts were prepared using a radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) according to the instructions of the manufacturer. The homogenate was centrifuged at 9168 g for 10 min at 4 °C. The supernatant was collected and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Solarbio Science & Technology Co., Ltd., Beijing, China). Total protein was denatured in 5×sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE) Sample Loading Buffer (Beyotime Biotechnology, Shanghai, China) at 100 °C. Equal amounts of proteins were separated by SDS-PAGE and then transferred to Immobilon®-P polyvinylidene difluoride (PVDF) transfer membranes (Merck Millipore Ltd., Massachusetts, USA). The membranes were blocked with 5% low fat dried milk and incubated with rabbit-derived monoclonal antibody to MMPs (MMP2, 1:1000; MMP7, 1:1000; MMP9, 1:1000; MMP13, 1:500; MMP14, 1:5000), TIMP-1 (1:250), and β-actin (1:1000), followed by goat-anti-rabbit-IgG coupled to horseradish peroxidase (1:10000). The bound antibody was detected using an enhanced chemiluminescence (ECL) detection system. β-Actin was used as a loading control. Densitometry analysis was performed using ImageJ 1.48v software (Wayne Rasband, National Institutes of Health, USA). Statistical analysis 2 The results were expressed as mean±standard deviation (SD). Data from the ELISA and western blot tests were analyzed using a one-way analysis of variance (ANOVA) with Duncan’s multiple range test and P values of < 0.05 were considered to be statistically significant. Results 1 Effects of Apios on body weight gain and food intake 2 Body weight and food intake were recorded during the experiment (Table 1 1 ). There were no significant changes in body weight among the different groups during the five days of the experiment. Moreover, Apios intervention did not affect the food intake of the rats. Model, post-partum rats on basal diets; Positive, post-partum rats on a basal diet plus 5.4 g/kg of Chanfukang granules; Low, post-partum rats on a basal diet containing 10% Apios powder; Medium, post-partum rats on a basal diet containing 20% Apios powder; High, post-partum rats on a basal diet containing 40% Apios powder. Control, non-pregnant rats on a basal diet. Values are presented as mean±SD ( n =6) Group Initial weight (g) Final weight (g) Food intake (g/d per rat) Model 393.2±42.3 412.5±24.0 40.1±5.1 Positive 410.5±31.3 420.5±13.4 42.2±5.5 Low 399.2±26.6 406.7±25.7 39.4±3.1 Medium 408.0±16.0 400.8±24.2 38.6±3.7 High 392.5±44.7 392.7±29.1 39.2±2.9 Control 401.7±18.8 407.7±28.1 39.1±1.3 Decreases in the uterus and spleen indices by Apios treatment 2 One of the most significant phenomena during pregnancy is the enlargement of the uterus. We determined the weight of uteri at post-partum Day 5 (Fig. 1a 1a ). The uterus indices of the model and positive groups were higher than that of the control group. However, low, medium, and high doses of Apios intervention significantly reduced the index, especially in the high dose Apios group. We also tested the spleen index (Fig. 1b 1b ). The spleen index of the model group was significantly higher than that of the control group. The spleen indices of the positive, low, medium, and high groups were lower than that of the model group, which indicated that Apios can reduce spleen enlargement caused by parturition. (a) Uterus index on post-partum Day 5. (b) Spleen index on post-partum Day 5. Both indices were calculated by dividing visceral weight measurements by the total rat weight. (c) Estrogen concentrations in fasting serum on post-partum Day 5. (d) Progesterone concentrations in fasting serum on post-partum Day 5. Model, post-partum rats on a basal diet; Positive, post-partum rats on a basal diet plus 5.4 g/kg of Chanfukang granules; Low, post-partum rats on a basal diet containing 10% Apios powder; Medium, post-partum rats on a basal diet containing 20% Apios powder; High, post-partum rats on a basal diet containing 40% Apios powder; Control, non-pregnant rats on a basal diet. Values are presented as mean±SD ( n =5 or 6 per group). Bars with different letters are significantly different among the experimental groups ( P < 0.05) Regulation of serum sex hormone concentrations by Apios treatment 2 To determine the effects of Apios on serum sex hormone concentrations in post-partum rats, we determined the estrogen and progesterone concentrations in serum. Estrogen secretion was higher in the low, medium, and high groups than in the model group (Fig. 1c 1c ). In contrast, progesterone secretion was lower in the low and medium groups (Fig. 1d 1d ). Though the two sex hormone concentrations showed different trends, the concentrations of both tended to return to the non-pregnant situation. Improvements and histological changes in the post-partum uterine horn by Apios treatment 2 We investigated the effect of Apios on tissue repair processes in post-partum uteri (Fig. 2 2 ). In the control group, uterine cavity morphology was normal, the endometrium was thick, and myocytes were arranged neatly (Figs. 2a and 2b 2a and 2b ). This was in contrast to tissue from of the model group in which the uterine cavity morphology was abnormal, the endometrium was thinner, and the arrangement of myocytes was irregular. Moreover, there was a large area of vascular hyalinization (Fig. 2a 2a ). After Apios intervention, uterine cavity morphology tended to be normal, the endometrium was thicker than that in the model group, and myocytes began to line up. In addition, the number of glands had increased and the level of vascular hyalinization had decreased. We also determined the thickness of the endometrium on lengthwise sections of uteri. After Apios intervention, the endometrium became thicker than that of the model group (Fig. 2c 2c ). (a) H & E staining on cross sections of the uterus on post-partum Day 5 (bar=500 μm). (b) H & E staining on lengthwise sections of the uterus on post-partum Day 5 (bar=1000 μm). (c) The thickness of the endometrium in lengthwise sections of the uterus on post-partum Day 5. The area of vascular hyalinization is indicated by the box. The areas of gland proliferation are indicated by arrows. The upper left corner of the thumbnail is an enlarged view of the location of the asterisk. Model, post-partum rats on a basal diet; Positive, post-partum rats on a basal diet plus 5.4 g/kg of Chanfukang granules; Low, post-partum rats on a basal diet containing 10% Apios powder; Medium, post-partum rats on a basal diet containing 20% Apios powder; High, post-partum rats on a basal diet containing 40% Apios powder; Control, non-pregnant rats on a basal diet. Values are presented as mean±SD ( n =4). Bars with different letters are significantly different among the experimental groups ( P < 0.05) Alleviation of inflammation by Apios treatment 2 To understand whether Apios treatment can alleviate the inflammatory response in uterine involution, we examined proinflammatory cytokines and immunoglobulins in serum. TNF-α, IL-1, IL-6, IgA, and IgG concentrations in the model group were significantly higher than those in the control group (Fig. 3 3 ). TNF-α secretion in the positive group was similar to that in the model group (Fig. 3a 3a ), but was down-regulated by Apios intervention. The same trends were observed for IL-1 (Fig. 3b 3b ) and IL-6 (Fig. 3c 3c ), which indicated that Apios can reduce the concentrations of proinflammatory cytokines. Serum concentrations of IgA (Fig. 3d 3d ) and IgG (Fig. 3e 3e ) in the Apios-treated group were lower than those in the model group, indicating that Apios can reduce the intensity of the immune response. (a) Tumor necrosis factor α (TNF-α) concentrations in fasting serum on post-partum Day 5. (b) Interleukin 1 (IL-1) concentrations in fasting serum on post-partum Day 5. (c) IL-6 concentrations in fasting serum on post-partum Day 5. (d) Immunoglobulin A (IgA) concentrations in fasting serum on post-partum Day 5. (e) IgG concentrations in fasting serum on post-partum Day 5. Model, post-partum rats on a basal diet; Positive, post-partum rats on a basal diet plus 5.4 g/kg of Chanfukang granules; Low, post-partum rats on a basal diet containing 10% Apios powder; Medium, post-partum rats on a basal diet containing 20% Apios powder; High, post-partum rats on a basal diet containing 40% Apios powder; Control, non-pregnant rats on a basal diet. Values are presented as mean±SD ( n =5 or 6). Bars with different letters are significantly different among the experimental groups ( P < 0.05) Regulation of transforming growth factor secretion by Apios treatment 2 To further explain the effect of Apios on the process of uterine involution, we tested the concentrations of transforming growth factors related to tissue repair. The serum concentrations of TGF-β1 in the Apios-treated groups were higher than that in the control group (Fig. 4a 4a ), but lower than that in the model group. However, the trends for TGF-β3 were opposite (Fig. 4b 4b ). After Apios intervention, a trend towards an increase in TGF-β3 secretion was observed. The ratio of TGF-β3 to TGF-β1 was also significantly increased by Apios treatment (Fig. 4c 4c ). (a) Transforming growth factor β1 (TGF-β1) concentrations in fasting serum on post-partum Day 5. (b) Transforming growth factor β3 (TGF-β3) concentrations in fasting serum on post-partum Day 5. (c) The ratio of TGF-β3 to TGF-β1 in serum levels on post-partum Day 5. Model, post-partum rats on a basal diet; Positive, post-partum rats on a basal diet plus 5.4 g/kg of Chanfukang granules; Low, post-partum rats on a basal diet containing 10% Apios powder; Medium, post-partum rats on a basal diet containing 20% Apios powder; High, post-partum rats on a basal diet containing 40% Apios powder; Control, non-pregnant rats on a basal diet. Values are presented as mean±SD ( n =5 or 6). Bars with different letters are significantly different among the experimental groups ( P < 0.05) Improvement in the levels of matrix metalloproteinases and inhibitors in post-partum uteri by Apios treatment 2 To elucidate the role of the degradation of collagen in post-partum uteri influenced by Apios, the expression of various MMPs and TIMP was analyzed semi-quantitatively by western blotting. The content of MMP9, MMP2, and proMMP2 in the Apios treatment groups was lower than that in the model group (Figs. 5b and 5c 5b and 5c ). The levels of MMP7, MMP13, and MMP14 in the Apios treatment groups were also lower than those in the model group (Figs. 5d–5f 5d–5f ). However, the level of TIMP-1 increased after Apios intervention (Fig. 5g 5g ). (a) Protein levels of MMP9, MMP2/proMMP2, MMP7, MMP13, MMP14, and TIMP-1 on post-partum Day 5. The intensity of bands corresponding to MMP9 (b), MMP2/proMMP2 (c), MMP7 (d), MMP13 (e), MMP14 (f), and TIMP-1 (g) in the uterine horn on post-partum Day 5 was corrected for β-actin (control). Model, post-partum rats on a basal diet; Low, post-partum rats on a basal diet containing 10% Apios powder; Medium, post-partum rats on a basal diet containing 20% Apios powder; High, post-partum rats on a basal diet containing 40% Apios powder. Values are presented as mean±SD ( n =5 or 6). Bars with different letters are significantly different among the experimental groups ( P < 0.05) Discussion 1 In women, the uterus is known to undergo physiological involution during a period of 6–8 weeks after delivery. Accelerating the involution process can be a way to avoid post-partum complications. In our study, we established an experimental animal model to prove our hypothesis that treatment with Apios can improve uterine involution. We found that Apios decreased uterus and spleen indices, and regulated sex hormones in post-partum rats. Moreover, Apios improved the morphological changes in post-partum uteri. The molecular mechanisms involved may include the alleviation of inflammation, the regulation of transforming growth factors, and a reduction in levels of MMPs. Several lines of evidence suggest that Apios may be a potential food for the improvement of uterine involution. Firstly, after Apios treatment, the degree of uterine remodeling was accompanied by an accelerated reduction in uterine weight and improvements in hormone levels. Uterine involution involves tissue destruction and subsequent repair (Alan and Liman, 2012 2012 ). Changes in the size of the uterus are the most intuitive indicator of the process. Apios treatment can accelerate a return to normal uterine weight, especially at high doses. Sex hormone levels, especially estrogen concentrations, are coincident with endometrial repair (Ferenczy et al., 1979 1979 ). In the rat, serum concentrations of estradiol and progesterone decrease rapidly after delivery, but begin to rise on Day 2 post-partum (Takamoto et al., 1998 1998 ). Our results showed that the serum concentrations of estrogen in the Apios treatment group were higher than that in the model group. Estrogens appear to be a major requirement for the growth of mammary ducts (Grota and Eik-Nes, 1967 1967 ). Therefore, Apios may help lactation. Grota and Eik-Nes ( 1967 1967 ) found a slight decrease in serum progesterone concentrations from Day 3 post-partum. Our results indicated that Apios intervention may help speed up this process (Fig. 1d 1d ). Secondly, Apios helped post-partum uterine reconstruction as shown by histology. After parturition, the endometrium and myometrium begin remodeling (Al-Bassam et al., 1981 1981 ; Gray et al., 2001 2001 ). The glands in the endometrium increase and smooth muscle cells begin to regain ordinary form and orientation (Henell et al., 1983 1983 ). Our results showed that Apios treatment increased the numbers of glands, thickened the endometrium, and promoted the orderly arrangement of muscle cells. In addition, in the sub-involuted uterus, vessels are often filled with red blood cells and thrombi, the walls of which show deposition of hyaline (Petrovitch et al., 2009 2009 ). Apios intervention could prevent this phenomenon (Fig. 2 2 ). Thirdly, Apios alleviated the secretion of proinflammatory cytokines. The uterus is sterile during the whole of pregnancy, but after parturition, microorganisms may easily contaminate the uterine lumen, as observed frequently in cattle (Griffin et al., 1974 1974 ; Sheldon et al., 2002 2002 ). From Day 0 (the parturition day) to Day 14, the number of uterine bacteria increased, and then the bacteria declined to normal levels within 60 d (Elliott et al., 1968 1968 ). During this period, contamination stimulated the immune response with phagocytes recruited to the uterine lumen, releasing proinflammatory cytokines, such as TNF-α, IL-1, and IL-6. These cytokines can cause an acute phase protein response to further increase phagocyte mobilization (Sheldon, 2004 2004 ). These changes indicated that these proinflammatory cytokines will decrease to normal levels when the uterine involution process is completed. Galvão et al. ( 2011 2011 ) found that from Week 1 to Week 7 post-partum, gene expression of proinflammatory cytokines (TNF-α, IL-1β) in the endometrium of normal cows decreased with time. IL-6 gene expression changed slightly. Kasimanickam et al. ( 2013 2013 ) found that serum concentrations of IL-1β decreased after one week and whereas IL-6 concentrations did not change significantly. However, TNF-α concentrations decreased during the first five weeks and increased at Week 7 in normal post-partum cows, which was contrary to these observed inflammatory conditions. This may be because cows in those groups had undergone body condition loss, causing an adipocyte-induced increase in TNF-α after five weeks. In our study, serum concentrations of TNF-α, IL-1, and IL-6 were lower in the Apios-treated groups than in the model group, which indicated that Apios intervention can inhibit pathogenic proliferation. Immunoglobulin concentrations in uterine secretions reflect the microbial challenge and the clinical recovery conditions in the endometrium (Azawi, 2008 2008 ). LeBlanc et al. ( 1988 1988 ) found that IgA and IgG concentrations in post-partum uterine fluids significantly decreased after one week. In the present study, we found that serum concentrations of IgA and IgG in the Apios intervention groups were lower than those in the model group, which also accounted for the alleviation of inflammation following Apios treatment. Fourthly, uterine tissue reconstructed following Apios treatment was associated with some peptides responsible for tissue repair. TGF-β is a pleiotropic cytokine belonging to the TGF-β superfamily that includes three different isoforms (TGF-β1–3) (O'kane and Ferguson, 1997 1997 ). TGF-β1 and TGF-β3 are important in stimulating wound contraction. It was reported that skin wounds without scars exhibited high concentrations of TGF-β3 and low concentrations of TGF-β1 (Lin et al., 1995 1995 ; Soo et al., 2003 2003 ), while an increased ratio of TGF-β1/β3 reduced scarring (O'kane and Ferguson, 1997 1997 ). Our results also showed that TGF-β1 and TGF-β3 were important biological factors involved in the uterine involution process following Apios intervention. Finally, Apios regulated the levels of MMPs and inhibitors in the post-partum uterus. After parturition, the involuting uterus goes through a rapid reduction in size which requires remodeling of the ECM, mainly the collagen (Curry and Osteen, 2003 2003 ). MMPs are Zn-dependent endopeptidases that can degrade most components of the ECM at neutral pH (Nagase et al., 2006 2006 ; Murphy and Nagase, 2008 2008 ). In addition to various types of collagens, MMP2 and MMP9 can cleave the gelation into small peptides (Shimizu et al, 1983 1983 ). These appear to increase on the day of labor, and then decrease 4 or 5 d after parturition (Manase et al., 2006 2006 ; Nguyen et al., 2016 2016 ). Our results showed that Apios treatment accelerated the reduction in MMP2 and MMP9 content in post-partum uteri. The activity of rat MMP7 was first identified in the post-partum uterus. MMP7 can degrade a wide range of gelatins, proteoglycans, and glycoproteins of the matrix (Woessner, 1996 1996 ). It was shown to reach a peak at 1–2 d after parturition, and then decrease (Sellers and Woessner, 1980 1980 ). The level of MMP7 was lower in the Apios treatment groups than in the model group (Fig. 5d 5d ). Collagenase activity reaches a maximum within 24 h and decreases slowly to low values at 5 d post-partum (Woessner, 1979 1979 ). In the rat uteri, we also detected MMP13, which is known to be orthologous to human interstitial collagenase-1 (Wolf et al., 1996 1996 ). We found that Apios accelerated the decline of MMP13. MMP14 causes the degradation of collagen types I, IV, and V, and is likely the activator of proMMP2 (Gaide Chevronnay et al., 2012 2012 ). Our results also showed that trends of proMMP2 content were similar to those of MMP14. In contrast to MMPs, TIMP-1 binds to almost all MMP active sites (Umenishi et al., 1991 1991 ; Willenbrock and Murphy, 1994 1994 ), preferring to bind to proMMP9 (Hulboy et al., 1997 1997 ). Apios treatment elevated the levels of TIMP-1 to inhibit all the MMPs (Fig. 5g 5g ). One limitation of the current study was that the addition of Apios to the diet may have led to an imbalance of energy or protein content in Apios groups. Though functional components of Apios helped uterine involution, protein availability or energy may play a role in the process. Conclusions 1 In summary, we have verified our hypothesis that post-partum recovery, particularly uterine involution, was improved by Apios treatment. We investigated hormone levels, morphological changes, and levels of various cytokines and enzymes, finding that Apios accelerated several processes related to uterine involution. Understanding the influence of Apios on the uterine healing process can provide direct evidence that it may be a potential functional food for post-partum females. Results from the current study suggest that the active ingredient in Apios is heat-stable. Hence, identifying the active ingredient and its properties would be important, and may provide clues as to its nature. References E Alan , N Liman . Immunohistochemical localization of beta defensins in the endometrium of rat uterus during the postpartum involution period . Vet Res Commun , 2012 . 36 ( 3 ): 173 - 185 . 22777508 DOI: 10.1007/s11259-012-9529-7 http://doi.org/10.1007/s11259-012-9529-7 . Immunohistochemical localization of beta defensins in the endometrium of rat uterus during the postpartum involution period MA Al-Bassam , RG Thomson , L O'Donnell . Normal postpartum involution of the uterus in the dog . Can J Comp Med , 1981 . 45 ( 3 ): 217 - 232 . 7340907 . Normal postpartum involution of the uterus in the dog L Alkema , D Chou , D Hogan , 等 . Global, regional, and national levels and trends in maternal mortality between 1990 and 2015, with scenario-based projections to 2030: a systematic analysis by the UN maternal mortality estimation inter-agency group . Lancet , 2016 . 387 ( 10017 ): 462 - 474 . 26584737 DOI: 10.1016/S0140-6736(15)00838-7 http://doi.org/10.1016/S0140-6736(15)00838-7 . Global, regional, and national levels and trends in maternal mortality between 1990 and 2015, with scenario-based projections to 2030: a systematic analysis by the UN maternal mortality estimation inter-agency group OI Azawi . Postpartum uterine infection in cattle . Anim Reprod Sci , 2008 . 105 ( 3-4 ): 187 - 208 . 18280065 DOI: 10.1016/j.anireprosci.2008.01.010 http://doi.org/10.1016/j.anireprosci.2008.01.010 . Postpartum uterine infection in cattle WT Chitisankul , K Shimada , Y Omizu , 等 . Mechanism of DDMP-saponin degradation and maltol production in soymilk preparation . LWT-Food Sci Technol , 2015 . 64 ( 1 ): 197 - 204 . DOI: 10.1016/j.lwt.2015.05.023 http://doi.org/10.1016/j.lwt.2015.05.023 . Mechanism of DDMP-saponin degradation and maltol production in soymilk preparation J Cui , Y Kim , EB Lee , 等 . Immunostimulatory activity of Apios tuber extract on RAW264.7 macrophage cells . J Bacteriol Virol , 2016 . 46 ( 4 ): 248 - 257 . DOI: 10.4167/jbv.2016.46.4.248 http://doi.org/10.4167/jbv.2016.46.4.248 . Immunostimulatory activity of Apios tuber extract on RAW264.7 macrophage cells Jr TE Curry , KG Osteen . The matrix metalloproteinase system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle . Endocr Rev , 2003 . 24 ( 4 ): 428 - 465 . 12920150 DOI: 10.1210/er.2002-0005 http://doi.org/10.1210/er.2002-0005 . The matrix metalloproteinase system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle L Elliott , KJ McMahon , HT Gier , 等 . Uterus of the cow after parturition: bacterial content . Am J Vet Res , 1968 . 29 ( 1 ): 77 - 81 . 5688806 . Uterus of the cow after parturition: bacterial content A Ferenczy , G Bertrand , MM Gelfand . Studies on the cytodynamics of human endometrial regeneration: III. In vitro short-term incubation historadioautography . Am J Obstet Gynecol , 1979 . 134 ( 3 ): 297 - 304 . 453263 DOI: 10.1016/S0002-9378(16)33036-8 http://doi.org/10.1016/S0002-9378(16)33036-8 . Studies on the cytodynamics of human endometrial regeneration: III. In vitro short-term incubation historadioautography HP Gaide Chevronnay , C Selvais , H Emonard , 等 . Regulation of matrix metalloproteinases activity studied in human endometrium as a paradigm of cyclic tissue breakdown and regeneration . Biochim Biophys Acta (BBA) Proteins Proteom , 2012 . 1824 ( 1 ): 146 - 156 . DOI: 10.1016/j.bbapap.2011.09.003 http://doi.org/10.1016/j.bbapap.2011.09.003 . Regulation of matrix metalloproteinases activity studied in human endometrium as a paradigm of cyclic tissue breakdown and regeneration KN Galvão , NR Santos , JS Galvão , 等 . Association between endometritis and endometrial cytokine expression in postpartum Holstein cows . Theriogenology , 2011 . 76 ( 2 ): 290 - 299 . 21496904 DOI: 10.1016/j.theriogenology.2011.02.006 http://doi.org/10.1016/j.theriogenology.2011.02.006 . Association between endometritis and endometrial cytokine expression in postpartum Holstein cows CA Gray , FF Bartol , BJ Tarleton , 等 . Developmental biology of uterine glands . Biol Reprod , 2001 . 65 ( 5 ): 1311 - 1323 . 11673245 DOI: 10.1095/biolreprod65.5.1311 http://doi.org/10.1095/biolreprod65.5.1311 . Developmental biology of uterine glands JF Griffin , PJ Hartigan , WR Nunn . Non-specific uterine infection and bovine fertility: I. Infection patterns and endometritis during the first seven weeks post-partum . Theriogenology , 1974 . 1 ( 3 ): 91 - 106 . 4619812 DOI: 10.1016/0093-691X(74)90052-1 http://doi.org/10.1016/0093-691X(74)90052-1 . Non-specific uterine infection and bovine fertility: I. Infection patterns and endometritis during the first seven weeks post-partum LJ Grota , KB Eik-Nes . Plasma progesterone concentrations during pregnancy and lactation in the rat . J Reprod Fertil , 1967 . 13 ( 1 ): 83 - 91 . 6066784 DOI: 10.1530/jrf.0.0130083 http://doi.org/10.1530/jrf.0.0130083 . Plasma progesterone concentrations during pregnancy and lactation in the rat WY Harianja , O Setiani , U Umaroh , 等 . The impact of pineapple ( Ananas comosus ( L .) Merr .) juice on fundal height in primigravida mothers during postpartum period . Belitung Nurs J , 2017 . 3 ( 2 ): 134 - 141 . . The impact of pineapple (Ananas comosus (L.) Merr.) juice on fundal height in primigravida mothers during postpartum period F Henell , JLE Ericsson , H Glaumann . An electron microscopic study of the post-partum involution of the rat uterus. With a note on apparent crinophagy of collagen . Virchows Arch B Cell Pathol Incl Mol Pathol , 1983 . 42 ( 1 ): 271 - 287 . 6134390 DOI: 10.1007/BF02890390 http://doi.org/10.1007/BF02890390 . An electron microscopic study of the post-partum involution of the rat uterus. With a note on apparent crinophagy of collagen K Hoshikawa , Juliarni . The growth of apios ( Apios americana Medikus), a new crop, under field conditions . Jpn J Crop Sci , 1995 . 64 ( 2 ): 323 - 327 . DOI: 10.1626/jcs.64.323 http://doi.org/10.1626/jcs.64.323 . The growth of apios (Apios americana Medikus), a new crop, under field conditions DL Hulboy , LA Rudolph , LM Matrisian . Matrix metalloproteinases as mediators of reproductive function . Mol Hum Reprod , 1997 . 3 ( 1 ): 27 - 45 . 9239706 DOI: 10.1093/molehr/3.1.27 http://doi.org/10.1093/molehr/3.1.27 . Matrix metalloproteinases as mediators of reproductive function RK Kasimanickam , VR Kasimanickam , JR Olsen , 等 . Associations among serum pro- and anti-inflammatory cytokines, metabolic mediators, body condition, and uterine disease in postpartum dairy cows . Reprod Biol Endocrinol, 11:103 , 2013 . DOI: 10.1186/1477-7827-11-103 http://doi.org/10.1186/1477-7827-11-103 . Associations among serum pro- and anti-inflammatory cytokines, metabolic mediators, body condition, and uterine disease in postpartum dairy cows J Kawamura , E Miura , K Kawakishi , 等 . Investigation of the safety and antihyperglycemic effect of Apios americana flower intake as a food material in normal and diabetic mice . Food Sci Technol Res , 2015 . 21 ( 3 ): 453 - 462 . DOI: 10.3136/fstr.21.453 http://doi.org/10.3136/fstr.21.453 . Investigation of the safety and antihyperglycemic effect of Apios americana flower intake as a food material in normal and diabetic mice C Kikuta , Y Sugimoto , Y Konishi , 等 . Physicochemical and structural properties of starch isolated from Apios americana Medikus . J Appl Glycosci , 2012 . 59 ( 1 ): 21 - 30 . DOI: 10.5458/jag.jag.JAG-2011_011 http://doi.org/10.5458/jag.jag.JAG-2011_011 . Physicochemical and structural properties of starch isolated from Apios americana Medikus H Kinugasa , Y Watanbe . Nutritional composition of the tubers of American groundnut ( Apios americana Medikus) . Sonoda Women’s Coll Stud , 1992 . 26 209 - 218 . . Nutritional composition of the tubers of American groundnut (Apios americana Medikus) HB Krishnan . Identification of genistein, an anticarcinogenic compound, in the edible tubers of the American groundnut ( Apios americana Medikus) . Crop Sci , 1998 . 38 ( 4 ): 1052 - 1056 . DOI: 10.2135/cropsci1998.0011183X003800040028x http://doi.org/10.2135/cropsci1998.0011183X003800040028x . Identification of genistein, an anticarcinogenic compound, in the edible tubers of the American groundnut (Apios americana Medikus) S Kuramoto , G Kaneyoshi , Y Morinaga , 等 . Angiotensin-converting enzyme-inhibitory peptides isolated from pepsin hydrolyzate of Apios americana tuber and their hypotensive effects in spontaneously hypertensive rats . Food Sci Technol Res , 2013 . 19 ( 3 ): 399 - 407 . DOI: 10.3136/fstr.19.399 http://doi.org/10.3136/fstr.19.399 . Angiotensin-converting enzyme-inhibitory peptides isolated from pepsin hydrolyzate of Apios americana tuber and their hypotensive effects in spontaneously hypertensive rats MM LeBlanc , PJ Hansen , WC Buhi . Uterine protein secretion in postpartum and cyclic mares . Theriogenology , 1988 . 29 ( 6 ): 1303 - 1316 . DOI: 10.1016/0093-691X(88)90010-6 http://doi.org/10.1016/0093-691X(88)90010-6 . Uterine protein secretion in postpartum and cyclic mares RY Lin , KM Sullivan , PA Argenta , 等 . Exogenous transforming growth factor-beta amplifies its own expression and induces scar formation in a model of human fetal skin repair . Ann Surg , 1995 . 222 ( 2 ): 146 - 154 . 7639582 DOI: 10.1097/00000658-199508000-00006 http://doi.org/10.1097/00000658-199508000-00006 . Exogenous transforming growth factor-beta amplifies its own expression and induces scar formation in a model of human fetal skin repair K Manase , T Endo , M Chida , 等 . Coordinated elevation of membrane type 1-matrix metalloproteinase and matrix metalloproteinase-2 expression in rat uterus during postpartum involution . Reprod Biol Endocrinol, 4:32 , 2006 . DOI: 10.1186/1477-7827-4-32 http://doi.org/10.1186/1477-7827-4-32 . Coordinated elevation of membrane type 1-matrix metalloproteinase and matrix metalloproteinase-2 expression in rat uterus during postpartum involution MR Morales , JG Foster . Forage potential of American potato bean . 2009 . Proceedings of the American Forage and Grassland Council. 2009 Annual Conference, Grand Rapids, Michigan . . Forage potential of American potato bean G Murphy , H Nagase . Progress in matrix metalloproteinase research . Mol Aspects Med , 2008 . 29 ( 5 ): 290 - 308 . 18619669 DOI: 10.1016/j.mam.2008.05.002 http://doi.org/10.1016/j.mam.2008.05.002 . Progress in matrix metalloproteinase research H Nagase , R Visse , G Murphy . Structure and function of matrix metalloproteinases and TIMPs . Cardiovasc Res , 2006 . 69 ( 3 ): 562 - 573 . 16405877 DOI: 10.1016/j.cardiores.2005.12.002 http://doi.org/10.1016/j.cardiores.2005.12.002 . Structure and function of matrix metalloproteinases and TIMPs K Nara , KI Nihei , Y Ogasawara , 等 . Novel isoflavone diglycoside in groundnut ( Apios americana Medik) . Food Chem , 2011 . 124 ( 3 ): 703 - 710 . DOI: 10.1016/j.foodchem.2010.05.107 http://doi.org/10.1016/j.foodchem.2010.05.107 . Novel isoflavone diglycoside in groundnut (Apios americana Medik) TTT Nguyen , O Shynlova , SJ Lye . Matrix metalloproteinase expression in the rat myometrium during pregnancy, term labor, and postpartum . Biol Reprod , 2016 . 95 ( 1 ): 24 27251092 DOI: 10.1095/biolreprod.115.138248 http://doi.org/10.1095/biolreprod.115.138248 . Matrix metalloproteinase expression in the rat myometrium during pregnancy, term labor, and postpartum S O'kane , MWJ Ferguson . Transforming growth factor βs and wound healing . Int J Biochem Cell Biol , 1997 . 29 ( 1 ): 63 - 78 . 9076942 DOI: 10.1016/S1357-2725(96)00120-3 http://doi.org/10.1016/S1357-2725(96)00120-3 . Transforming growth factor βs and wound healing I Petrovitch , RB Jeffrey , A Heerema-McKenney . Subinvolution of the placental site . J Ultrasound Med , 2009 . 28 ( 8 ): 1115 - 1119 . 19643799 DOI: 10.7863/jum.2009.28.8.1115 http://doi.org/10.7863/jum.2009.28.8.1115 . Subinvolution of the placental site B Reynolds , W Blackmon , E Wickremesinhe , 等 . Janick J . Domestication of Apios americana . 1988 . Advances in New Crops: Proceedings of the First National Symposium New Crops: Research, Development, Economics, Timber Press , 436 - 442 . . Domestication of Apios americana T Rosen . Placenta accreta and cesarean scar pregnancy: overlooked costs of the rising cesarean section rate . Clin Perinatol , 2008 . 35 ( 3 ): 519 - 529 . 18952019 DOI: 10.1016/j.clp.2008.07.003 http://doi.org/10.1016/j.clp.2008.07.003 . Placenta accreta and cesarean scar pregnancy: overlooked costs of the rising cesarean section rate LA Salamonsen . Tissue injury and repair in the female human reproductive tract . Reproduction , 2003 . 125 ( 3 ): 301 - 311 . 12611594 DOI: 10.1530/rep.0.1250301 http://doi.org/10.1530/rep.0.1250301 . Tissue injury and repair in the female human reproductive tract A Sellers , Jr JF Woessner . The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan . Biochem J , 1980 . 189 ( 3 ): 521 - 531 . 7011317 DOI: 10.1042/bj1890521 http://doi.org/10.1042/bj1890521 . The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan IM Sheldon . The postpartum uterus . Vet Clin North Am Food Anim Pract , 2004 . 20 ( 3 ): 569 - 591 . 15471625 DOI: 10.1016/j.cvfa.2004.06.008 http://doi.org/10.1016/j.cvfa.2004.06.008 . The postpartum uterus IM Sheldon , DE Noakes , AN Rycroft , 等 . Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle . Reproduction , 2002 . 123 ( 6 ): 837 - 845 . 12052238 DOI: 10.1530/rep.0.1230837 http://doi.org/10.1530/rep.0.1230837 . Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle K Shimizu , T Furuya , Y Takeo , 等 . Clearance of materials from breakdown of uterine collagen in mice during postpartum involution . Acta Anat (Basel) , 1983 . 116 ( 1 ): 10 - 13 . 6683059 DOI: 10.1159/000145720 http://doi.org/10.1159/000145720 . Clearance of materials from breakdown of uterine collagen in mice during postpartum involution VA Shkurupiy , KS Obedinskaya , AP Nadeev . Morphological study of the main mechanisms of myometrium involution after repeated pregnancies in mice . Bull Exp Biol Med , 2011 . 150 ( 3 ): 378 - 382 . 21240359 DOI: 10.1007/s10517-011-1147-9 http://doi.org/10.1007/s10517-011-1147-9 . Morphological study of the main mechanisms of myometrium involution after repeated pregnancies in mice C Soo , SR Beanes , FY Hu , 等 . Ontogenetic transition in fetal wound transforming growth factor-β regulation correlates with collagen organization . Am J Pathol , 2003 . 163 ( 6 ): 2459 - 2476 . 14633618 DOI: 10.1016/S0002-9440(10)63601-2 http://doi.org/10.1016/S0002-9440(10)63601-2 . Ontogenetic transition in fetal wound transforming growth factor-β regulation correlates with collagen organization N Takamoto , PC Leppert , SY Yu . Cell death and proliferation and its relation to collagen degradation in uterine involution of rat . Connec Tissue Res , 1998 . 37 ( 3-4 ): 163 - 175 . DOI: 10.3109/03008209809002436 http://doi.org/10.3109/03008209809002436 . Cell death and proliferation and its relation to collagen degradation in uterine involution of rat M Takashima , K Nara , E Niki , 等 . Evaluation of biological activities of a groundnut ( Apios americana Medik) extract containing a novel isoflavone . Food Chem , 2013 . 138 ( 1 ): 298 - 305 . 23265491 DOI: 10.1016/j.foodchem.2012.10.100 http://doi.org/10.1016/j.foodchem.2012.10.100 . Evaluation of biological activities of a groundnut (Apios americana Medik) extract containing a novel isoflavone F Umenishi , M Umeda , K Miyazaki . Efficient purification of TIMP-2 from culture medium conditioned by human hepatoma cell line, and its inhibitory effects on metalloproteinases and in vitro tumor invasion . J Biochem , 1991 . 110 ( 2 ): 189 - 195 . 1662201 DOI: 10.1093/oxfordjournals.jbchem.a123555 http://doi.org/10.1093/oxfordjournals.jbchem.a123555 . Efficient purification of TIMP-2 from culture medium conditioned by human hepatoma cell line, and its inhibitory effects on metalloproteinases and in vitro tumor invasion QH Wang , S Zhang , LM Qin , 等 . Yimu San improves obstetric ability of pregnant mice by increasing serum oxytocin levels and connexin 43 expression in uterine smooth muscle . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2017 . 18 ( 11 ): 986 - 993 . DOI: 10.1631/jzus.B1600289 http://doi.org/10.1631/jzus.B1600289 . Yimu San improves obstetric ability of pregnant mice by increasing serum oxytocin levels and connexin 43 expression in uterine smooth muscle United Nations Population Fund WHO , UNICEF . Childbirth: labour, delivery and immediate postpartum care . Pregnancy, Childbirth, Postpartum and Newborn Care: A Guide for Essential Practice, 3rd Ed , 2015 . World Health Organization, Geneva , 85 - 88 . . Childbirth: labour, delivery and immediate postpartum care Maternal mortality . 2018 . http://www.who.int/en/news-room/fact-sheets/detail/maternal-mortality [Accessed on June 4, 2018] . . Maternal mortality F Willenbrock , G Murphy . Structure-function relationships in the tissue inhibitors of metalloproteinases . Am J Respir Crit Care Med , 1994 . 150 ( 6 Pt 2 ): S165 - S170 . 7952654 DOI: 10.1164/ajrccm/150.6_Pt_2.S165 http://doi.org/10.1164/ajrccm/150.6_Pt_2.S165 . Structure-function relationships in the tissue inhibitors of metalloproteinases PW Wilson , JR Gorny , WJ Blackmon , 等 . Fatty acids in the American groundnut ( Apios americana ) . J Food Sci , 1986 . 51 ( 5 ): 1387 - 1388 . DOI: 10.1111/j.1365-2621.1986.tb13136.x http://doi.org/10.1111/j.1365-2621.1986.tb13136.x . Fatty acids in the American groundnut (Apios americana) Jr JF Woessner . Total, latent and active collagenase during the course of post-partum involution of the rat uterus. Effect of oestradiol . Biochem J , 1979 . 180 ( 1 ): 95 - 102 . 226069 DOI: 10.1042/bj1800095 http://doi.org/10.1042/bj1800095 . Total, latent and active collagenase during the course of post-partum involution of the rat uterus. Effect of oestradiol Jr JF Woessner . Regulation of matrilysin in the rat uterus . Biochem Cell Biol , 1996 . 74 ( 6 ): 777 - 784 . 9164647 DOI: 10.1139/o96-084 http://doi.org/10.1139/o96-084 . Regulation of matrilysin in the rat uterus K Wolf , P Sandner , A Kurtz , 等 . Messenger ribonucleic acid levels of collagenase (MMP-13) and matrilysin (MMP-7) in virgin, pregnant, and postpartum uterus and cervix of rat . Endocrinology , 1996 . 137 ( 12 ): 5429 - 5434 . 8940367 DOI: 10.1210/endo.137.12.8940367 http://doi.org/10.1210/endo.137.12.8940367 . Messenger ribonucleic acid levels of collagenase (MMP-13) and matrilysin (MMP-7) in virgin, pregnant, and postpartum uterus and cervix of rat FJ Yan , YY Yang , LS Yu , 等 . Effects of C -glycosides from Apios americana leaves against oxidative stress during hyperglycemia through regulating mitogen-activated protein kinases and nuclear factor erythroid 2-related factor 2 . J Agric Food Chem , 2017 . 65 ( 34 ): 7457 - 7466 . 28758742 DOI: 10.1021/acs.jafc.7b03163 http://doi.org/10.1021/acs.jafc.7b03163 . Effects of C-glycosides from Apios americana leaves against oxidative stress during hyperglycemia through regulating mitogen-activated protein kinases and nuclear factor erythroid 2-related factor 2 A Yoshii , S Kitahara , H Ueta , 等 . Role of uterine contraction in regeneration of the murine postpartum endometrium . Biol Reprod , 2014 . 91 ( 2 ): 32 24966392 DOI: 10.1095/biolreprod.114.117929 http://doi.org/10.1095/biolreprod.114.117929 . Role of uterine contraction in regeneration of the murine postpartum endometrium YZ Zhang , CS Zhou , SM Tang , 等 . Effect of AATI, a bowman-birk type inhibitor from Apios americana , on proliferation of cancer cell lines . Food Chem , 2011 . 128 ( 4 ): 909 - 915 . DOI: 10.1016/j.foodchem.2011.03.117 http://doi.org/10.1016/j.foodchem.2011.03.117 . Effect of AATI, a bowman-birk type inhibitor from Apios americana, on proliferation of cancer cell lines Compliance with ethics guidelines Zi-huan ZHENG, Ying HAN, Shi-ying YOU, Zuo CHEN, and Xiao-dong ZHENG declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1800475 Project supported by the Zhejiang Provincial Natural Science Foundation of China (No. LZ14C200001) 2018-09-12 2019-02-06 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 20