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Understanding and modeling channels of high-pressure liquids during hydraulic fracturing and stimulated reservoir volume (SRV) estimation are critical for the evaluation of the fracturing effect and the prediction of oil and gas production in unconventional (low permeability and porosity) reservoirs. In this study, we developed a “focal mechanism-to-event” channel connection criterion based on the timing, location, and focal mechanism of microseismic events, enabling the channel to be perpendicular or parallel to the fracturing plane and more in line with the mechanical properties of rock rupture. We first evaluated the two endpoints of all channels as a set of spatial points, followed by the application of the alpha-shape method to detect the concave hull shape boundary formed by the spatial points. Finally, we calculated the SRV by combining the shapes of the concave hull and Delaunay triangulation. In the experimental simulation, we found that this method could effectively calculate the hull volume, and the detected concave hull included all microseismic events and channels. Ultimately, we applied the channel connection criterion and SRV estimation method to a stage of 3.18 h real fracturing data containing 216 microseismic events. The final calculation revealed a total of 410 connection channels and 4.3154 × 105 m3 SRV, which is more consistent with the expected than the “event-to-event” or “event-to-channel” connection criteria and bin or convex hull SRV calculation methods.

Background Oxaliplatin resistance is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy and the risks in relapse and metastasis. However, the mechanism of Oxaliplatin resistance has not been completely elucidated. Methods Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10 transporter. The Cancer Genome Atlas data was analyzed to figure out the correlation between the clinical manifestation and ABCC10 expression. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 assay was conducted to identify the CRC cell viability and Oxaliplatin IC50. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Results CRC patients with higher ABCC10 were prone to relapse and metastasis. Differential ABCC10 expression in multiple CRC cell lines revealed a strong positive correlation between ABCC10 expression level and Oxaliplatin resistance. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was evidently elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. To explore a strategy to block ABCC10 in CRC cells, we paid a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that neither the inhibition of ERS nor the induction of mild ERS had anti-CRC effect. However, the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the induction of intense ERS. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS down-regulated ABCC10 expression via regulated IRE1-dependent decay activity. Conclusion Oxaliplatin was a substrate of ABCC10 efflux transporter. The intense ERS/IRE1α alleviated Oxaliplatin resistance through down-regulating ABCC10 in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Purpose: Colorectal cancer (CRC) ranks 2nd in morbidity and 3rd in mortality among all cancers worldwide. 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) is ubiquitously expressed in humans. Remarkably, the mevalonate pathway is often dysregulated in many cancers, suggesting tumor dependency on this classic metabolic pathway. In addition to altered substrates in the mevalonate pathway, it was recently found that HMGCS1 was highly expressed in prostate, melanoma and breast cancers. The transmembrane glycoprotein KIT is critical for the survival and development of various cells through activating downstream pathways and targets. Our previous research has proved that KIT is hyper-expressed in CRC and promotes CRC progression. However, whether HMGCS1 mediates the tumor-promoting effect of KIT signaling has not been unclosed.Material and Methods: We investigated the regulatory mechanism of KIT signaling on HMGCS1 expression and the role of increased HMGCS1 in CRC development by the use of gene over-expression and knock-down techniques in CRC cells, biological function tests, protein-DNA binding detection, bioinformatics, database analysis and c-kit loss-of-function mutant mice (Wadsm/m). Results: HMGCS1 was up-regulated by KIT-JNK-c-Jun signaling in CRC cells and could be a biomarker for CRC. Highly expressed HMCGS1 promoted CRC cell proliferation and invasion while inhibiting apoptosis. Our results provided evidence for the role of HMGCS1 in CRC progression and suggested that blocking KIT-JNK-c-Jun-HMGCS1 might be a new strategy for treating CRC patients.Conclusion: Our results indicated that KIT signaling could up-regulate HMGCS1 in CRC mediated by Ap-1/c-Jun. Hyper-expressed HMGCS1 promotes CRC cell proliferation and invasion while inhibiting apoptosis.

Background: Oxaliplatin resistance is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy. However, the mechanism of Oxaliplatin resistance has not been completely elucidated. In this study, we explored the key molecule involved in the Oxaliplatin resistance, which could be a candidate therapeutic target to attenuate chemo-resistance in CRC cells.Methods: Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10. The Oncomine Oncology Database and the Cancer Genome Atlas (TCGA) data were analyzed to figure out the correlation between the clinical manifestation and ABCC10. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 (CCK-8) assay was conducted to identify the CRC cell viability and Oxaliplatin IC50. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS).Results: ABCC10 transporter was correlated with CRC relapse and metastasis, indicating a CRC-promoting effect of ABCC10. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was significantly elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. We next explored a strategy to inhibit ABCC10 in CRC cells, paying a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the intense ERS/UPR instead of the inhibited and mild ERS/UPR. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS/UPR down-regulated ABCC10 via regulated IRE1-dependent decay (RIDD) activity. Conclusion: Oxaliplatin was a substrate of ABCC10. The intense ERS/IRE1α elicited anti-CRC effects through down-regulating ABCC10 so as to increase Oxaliplatin sensitivity, in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Zika virus (ZIKV) belongs to mosquito-borne flaviviruses. Unlike other members in the family, ZIKV can be sexually transmitted, and the female genital tracts are susceptible to ZIKV. However, the impacts of ZIKV infection on nonpregnant female reproductive health are not understood. In this study, we investigated the effects of ZIKV infection on the ovary by using nonpregnant female interferon α/β receptor-deficient (Ifnar1-/-) mice. The results showed that the ovary supported ZIKV replication, and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly reduced the numbers of antral follicles, aggravated follicular atresia and disrupted folliculogenesis. Notably, ZIKV replication in the ovary caused disordered ovarian steroidogenesis manifested by decreased expression of key enzymes linked to sex hormone synthesis including the cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1). Further, we observed that ZIKV infection disrupted the estrous cycle, and thus prolonged the time to conceive. More importantly, although ZIKV RNA could not be detected at 3 months post infection, the damaged ovarian structure and dysfunction were also observed. Taken together, our study demonstrates that ZIKV infection in nonpregnant female mice cause ovarian damage and dysfunction, even long after ZIKV clearance. These data provide important information to understand the effects of ZIKV infection in female reproductive tissues and basic evidence for further studies. IMPORTANCE ZIKV, a flavivirus, is primarily transmitted by mosquito bites. But it can also be transmitted vertically and sexually. Although ZIKV-associated Guillain-Barre syndrome and microcephaly have drawn great attention, there have been few studies on the potential effects of ZIKV on genital tract of non-pregnant female. This study investigated the effects of ZIKV on the ovary in mice. We found that ZIKV replicated in the ovary and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly damaged ovarian structure and function, and disrupted folliculogenesis. Notably, ZIKV infection further disrupted the estrous cycle and prolonged the time to conceive in mice by causing disordered ovarian steroidogenesis. These effects were observed in both the acute phase and the recovery phase after viral elimination. Overall, the new findings provide important additions to make out the potential adverse impacts of ZIKV on reproductive health in females.

Background and Aims Disruption of the intestinal barrier of the digestive tract is a common pathophysiological change in the elderly, which may partly contribute to gut dysfunction and inflammatory bowel disease [IBD]. This study aimed to discover new interactive epigenetic regulation patterns involved in intestinal barrier dysfunction and colitis in elderly populations. Methods Intestinal barrier function and structure were evaluated in naturally ageing mice and elderly people. High-throughput analysis was performed on colonic tissues from humans and mice. The synergistic roles of miR-1-3p and miR-124-3p were identified using microRNA mimic/agomirs. Related genes were examined in biopsies of old IBD patients. Results A defective mucus barrier was observed before mucosal microstructural damage during ageing. Elevated miR-1-3p expression in the colons of older individuals impaired the mucus barrier by directly targeting T-synthase, similarly to the mechanism of miR-124-3p, which we reported previously. Importantly, the synergistic effect of a half dose of each microRNA supplement on T-synthase and CDK4/6 was stronger than that of a full dose of miR-1-3p or miR-124-3p alone, and mice co-treated with two microRNAs showed greater susceptibility to chemical-induced colitis than mice treated with either microRNA alone. These two microRNAs were up-expressed in old IBD patients. Conclusions The slight increases in miR-1-3p and miR-124-3p expression with ageing may be important contributors to the breakdown of intestinal homeostasis by targeting divergent genes in different cells. These data reveal the potential ability of multiple microRNAs to exert synergistic effects to damage the intestinal barrier and promote inflammatory bowel disease development in elderly populations.

Aging is a significant risk factor for gastrointestinal dysmotility, but aging-associated differences between different organs and the exact time to start degenerating have remained obscure. Here we evaluated alterations of interstitial cells of Cajal, enteric neurons and connexin43 expression in the stomach, jejunum and colon in 2-, 12-, 16-, 20- and 24-month-old mice, as well as in aged human colon. Interstitial cells of Cajal, cholinergic and nitrergic neurons within the whole digestive tract were reduced over time, but their loss first appeared in stomach, then in intestine, helping to understand that gastric function was first impaired during aging. The decrease of connexin43 expression occurred before interstitial cells of Cajal and neurons loss, suggesting that connexin43 might be the major target influenced during senescence. Furthermore, changes in expressions of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin-1β, interleukin-6) and apoptosis-related proteins (B-cell lymphoma-2, caspase-3) which indicated “inflammaging”, might contribute to the loss of enteric neurons and interstitial cells of Cajal in aged gastrointestinal tract. Our results provide possible therapeutic time window for beneficial intervention for geriatric patients with gastrointestinal motility disorders.

The risk of colitis and colorectal cancer increases markedly throughout adult life, endangering the health and lives of elderly individuals. Previous studies have proposed that bacterial translocation and infection are the main risk factors for these diseases. Therefore, in the present study, we aimed to identify the underlying mechanism by focusing on the mucus barrier function and mucin‐type O‐glycosylation. We evaluated alterations in the colon mucus layer in 2‐, 16‐, and 24‐month‐old mice and aged humans. Aged colons showed defective intestinal mucosal barrier and changed mucus properties. The miR‐124‐3p expression level was significantly increased in the aged distal colonic mucosa, which was accompanied by an increase in pathogens and bacterial translocation. Meanwhile, T‐synthase, the rate‐limiting enzyme in O‐glycosylation, displayed an age‐related decline in protein expression. Further experiments indicated that miR‐124‐3p modulated O‐glycosylation by directly targeting T‐synthase. Moreover, young mice overexpressing miR‐124‐3p exhibited abnormal glycosylation, early‐onset, and more severe colitis. These data suggest that miR‐124‐3p predisposes to senile colitis by reducing T‐synthase, and the miR‐124‐3p/T‐synthase/O‐glycans axis plays an essential role in maintaining the physiochemical properties of colonic mucus and intestinal homeostasis., Young colons show intact inner layer and mucin‐type O‐glycosylation, limiting microbiota access to the epithelium. Age‐related alteration in miR‐124‐3p/T‐synthase/O‐glycans axis and reduction in mucus thickness weakened the mucus barrier, leading to abnormal bacterial intrusion and increased susceptibility to senile colitis.

Mesenchymal stem cells (MSCs) are considered a promising candidate for use in cell-based therapy for cartilage repair. To promote understanding of the molecular control of chondrogenesis differentiation in MSCs, we compared the changes in microRNAs during in vitro chondrogenesis process of human bone-marrow mesenchymal stem cells (hBMSCs). MiR-199b-5p was up-regulated significantly during this process. The aim of the study was to investigate the effects of miR-199b-5p on chondrogenic differentiation of C3H10T1/2 MSC cells and explore the underlying mechanisms. MiR-199b-5p mimics or inhibitor were transfected into C3H10T1/2 cells, respectively, and then, the effects of miR-199b-5p on chondrogenic differentiation of C3H10T1/2 cells were detected. The results indicated that miR-199b-5p overexpression inhibited the growth of C3H10T1/2 cells but promoted transforming growth factor-β3 (TGF-β3)-induced C3H10T1/2 cells of chondrogenic differentiation, as supported by enhancing the gene and protein expression of chondrocyte specific markers of SOX9, aggrecan, and collagen type II (Col2a1). In contrast, inhibiting miR-199b-5p notably promoted the proliferation of C3H10T1/2 cells but decreased chondrogenic differentiation. Furthermore, mechanism studies revealed that JAG1 was a direct target of miR-199b-5p by dual luciferase reporter assays. While silencing of JAG1 by isRNA resulted an increase of chondrogenic differentiation. Further, JAG1 knockdown was demonstrated to block the effect of miR-199b-5p inhibition. In conclusion, the present study revealed for the first time that miR-199b-5p was the positive regulators to modulate chondrogenic differentiation of C3H10T1/2 cells by targeting JAG1. These findings may provide a novel insight on miRNA-mediated MSC therapy for cartilage related disorders.

Low expression of the tumor suppressor Kelch-like ECH-associated protein 1 (KEAP1) in non–small cell lung cancer (NSCLC) often results in higher malignant biological behavior and poor prognosis; however, the underlying mechanism remains unclear. The present study demonstrates that overexpression of Keap1 significantly suppresses migration and invasion of three different lung cancer cells (A549, H460, and H1299). Highly expressed Keap1, compared with the control, promotes formation of multiple stress fibers with larger mature focal adhesion complexes in the cytoplasm where only fine focal adhesions were observed in the membrane under control conditions. RhoA activity significantly increased when Keap1 was overexpressed, whereas Myosin 9b expression was reduced but could be rescued by proteasome inhibition. Noticeably, mouse tumor xenografts with Keap1 overexpression were smaller in size and less metastatic relative to the control group. Taken together, these results demonstrate that Keap1 stabilizes F-actin cytoskeleton structures and inhibits focal adhesion turnover, thereby restraining the migration and invasion of NSCLC. Therefore, increasing Keap1 or targeting its downstream molecules might provide potential therapeutic benefits for the treatment of patients with NSCLC. Implications: This study provides mechanistic insight on the metastatic process in NSCLC and suggests that Keap1 and its downstream molecules may be valuable drug targets for NSCLC patients. Mol Cancer Res; 16(3); 508–16. ©2018 AACR.

For the first time, the composition, concentration and distribution characteristics of typical lipophilic marine algal toxins (LMATs) are investigated in surface seawater, suspended particulate matter (SPM) and sediments from the Yellow Sea and the Bohai Sea, China. Pectenotoxin-2 (PTX2) and okadaic acid (OA) were detected in offshore surface seawater samples (n=67) of the Yellow and Bohai Seas, and PTX2 was found in higher concentrations than OA. The concentrations of PTX2 were between 0.49 and 14.14ng/L. OA, dinophysistoxin-1(DTX1), PTX2 and gymnodimine (GYM) were detected in the nearshore surface seawater samples (n=20) of the Haizhou Bay of the Yellow Sea. OA concentrations were between 11.47 and 55.85ng/L. There was a large degree of variation in the concentrations of DTX1, fromlimit of quantification (LOQ) up to 143.14ng/L. The nearshore surface seawater samples (n=13) obtained from the Longdao area of the Bohai Sea mainly contain OA (5.00-24.14ng/L) and DTX1 (1.52-49.39ng/L), with PTX2 concentrations being less than the LOQ. Several LMATs were also detected both in the SPM and sediment samples of each study area. In summary, the LMAT composition and concentration levels in the surface seawater found in nearshore samples were substantially different from those of samples from the distant shore, which indicated the obvious regional characteristics of LMATs according to their spatial distribution in the Yellow Sea and the Bohai Sea.

Macrophage receptor with collagenous structure (MARCO) is a member of the class A scavenger receptor family which is expressed on the cell surface of macrophages. It is well known that MARCO avidly binds to unopsonized pathogens, leading to its ingestion by macrophages. However, the role of MARCO in the recognition and phagocytosis of tumor cells by macrophages remains poorly understood. In this study, we used lentiviral technology to knockdown and overexpress MARCO and investigated the ability of macrophages to phagocytose tumor cells. Our results showed that MARCO expression was correlated with the ability of macrophages to carry on phagocytosis. MARCO knockdown led to significant decreases in the number of engulfment pseudopodia of macrophages and inhibition of the phagocytosis of tumor cells. On the other hand, MARCO overexpression elevated activity of SYK, PI3K and Rac1 in macrophages, which led to changes in macrophage morphology and enhanced phagocytosis by promoting formation of stress fibers and pseudopodia. By Co-IP analysis we showed that MARCO directly binds to the β5 integrin of SL4 tumor cells. In summary, these results demonstrated the important role for MARCO in demonstrated tumor cells uptake and clearance by macrophages.

Autophagy plays a key role in pulmonary vascular remodeling via regulation of apoptosis and hyperproliferation of pulmonary arterial endothelial cells, which are the subject of increased attention. Autophagy-related 101 (ATG101) is an essential gene for the initiation of autophagy. Although the structure of ATG101 has been well-characterized, its exact biological function in autophagy is still unknown. In this study, an ATG101 single-stranded antisense RNA-loaded DNA triangular nanoparticle (ssATG101-TNP) is constructed to knock down the ATG101 gene expression. ssATG101-TNP can be effectively transfected into human pulmonary arterial endothelial cells (HPAECs) in time- and dose-dependent manners. Knockdown of ATG101 promotes cell apoptosis as well as inhibits cell autophagy and proliferation with hypoxic stimulation. Additionally, the hedgehog/Gli signal pathway is involved in ATG101-mediated macroautophagy and HPAEC proliferation. This study found that ATG101, an important member of the autophagy gene family, can regulate cell macroautophagy, apoptosis, and growth in HPAECs. ssATG101-TNP is demonstrated to be a nontoxic, highly efficient, gene-delivery vehicle for HPAECs. These findings also suggest that ATG101 might be a potential therapeutic target in diseases involving endothelial injury.

Gastrointestinal motility disorders (GMDs) are attributed to loss of interstitial cells of Cajal (ICC), whose survival and function are deeply dependent on the activation of KIT/SCF signalling. Based on the facts that gastrointestinal distention is common in GMD patients and SCF produced by smooth muscle cells (SMCs) is usually decreased before ICC loss, we considered a possible contribution of persistent gastrointestinal distention/stretch to SCF deficiency. In this study, chronic colonic distention mouse model, diabetic gastrointestinal paresis mouse model, cultured mouse colonic SMCs and colon specimens from Hirschsprung's disease patients were used. The results showed that SCF was clearly decreased in distent colon of mice and patients, and microRNA array and real‐time PCR indicated a concomitant increase of miR‐34c in distent colon. A negative regulation of miR‐34c on SCF expression was confirmed by luciferase reporter assays together with knock‐down and overexpression of miR‐34c in cultured colonic SMCs. Using EMSA and ChIP assays, we further consolidated that in response to persistent stretch, the transcription factor AP‐1/c‐Jun was highly activated in colonic SMCs and significantly promoted miR‐34c transcription by binding to miR‐34c promoter. Knock‐down or overexpression of AP‐1/c‐Jun in cultured colonic SMCs leads to down‐ or up‐regulation of miR‐34c, respectively. In addition, the activation of AP‐1/c‐Jun was through ERK1/2 signalling provoked by Ca2+ overload in colonic SMCs that were subject to persistent stretch. In conclusion, our data demonstrated that persistent distention/stretch on colonic SMCs could suppress SCF production probably through Ca2+‐ERK‐AP‐1‐miR‐34c deregulation, resulting in ICC loss or impairment and GMD progress.

It was reported that Src-mediated and RTK-dependent accumulation of key transcription factor, ETV4, which played an important role in the migration of embryonic cells and tumor cells, were regulated by their common downstream MAPK molecules. However, the detailed mechanism was not completely clear. In the present study, we revealed that ETV4 protein was significantly enhanced by ERK kinase activation in the colorectal cancer (CRC) patients and mouse models as well as in the CRC cell lines. It was further confirmed that the activation of ERK kinase led to the phosphorylation of ETV4 at Ser73 and the ETV4 phosphorylation could block its binding to COP1, thereby stabilized ETV4 via avoiding its ubiquitination degradation. In addition, this effect was not due to altering an E3 ubiquitin ligase, COP1 amount or p-COP1/COP1 ratio. Our results will help understand the mechanism of ETV4 overexpression in CRC patients and provide a clue to search new therapeutic target to treat the related tumors in clinical practice.

Acupuncture is a therapy effective in treating postoperative ileus (POI); its underlying mechanisms remain unclear. MicroRNAs (miRNAs) participate in inflammation and injury to the interstitial cells of Cajal (ICCs), both of which are considered to be contributors to POI. C-kit, encoding KIT, a specific marker of ICCs, is predicted to be targeted by miR-19a, an inflammation-related miRNA. Therefore, we investigated a possible link between inflammation, miR-19a, and ICCs in POI, as well as the mechanism by which these factors are affected by acupuncture. The effects of acupuncture on POI were assessed in patients after colorectal resection and in colocolic anastomosis mice. Immunofluorescence staining demonstrated that KIT[Formula: see text]/ano1[Formula: see text] ICCs dramatically decreased around the colonic incision in mice, which was negatively correlated with the pronounced increase in macrophage. However, this decrease was not due to apoptosis. IL-6R was expressed in ICCs, and IL-6 level was significantly increased, as measured by ELISA, in accompaniment with high miR-19a expression. The increase in IL-6 and miR-19a levels was negatively correlated with the decrease in KIT[Formula: see text]/ano1[Formula: see text] ICCs. A luciferase reporter assay demonstrated that miR-19a directly targeted C-kit, indicating that miR-19a caused ICC damage. Interestingly, acupuncture inhibited macrophage activation, IL-6 release, and miR-19a upregulation, while promoting KIT and ano1 restoration in ICCs. High serum miR-19a level in patients after colorectal resection was also reduced by acupuncture. Conclusively, the IL-6 released by macrophages during gastrointestinal surgery upregulated miR-19a, which downregulated KIT in ICCs and finally resulted in POI. Acupuncture can interfere with the “IL-6–miR-19a–KIT” axis, suggesting that it may be a therapeutic mechanism that works against POI.

Gastrointestinal (GI) distention is a common pathological characteristic in most GI motility disorders (GMDs), however, their detail mechanism remains unknown. In this study, we focused on Ca2+ overload of smooth muscle, which is an early intracellular reaction to stretch, and its downstream MAPK signaling and also reduction of SCF in vivo and in vitro. We successfully established colonic dilation mouse model by keeping incomplete colon obstruction for 8 days. The results showed that persistent colonic dilation clearly induced Ca2+ overload and activated all the three MAPK family members including JNK, ERK and p38 in smooth muscle tissues. Similar results were obtained from dilated colon of patients with Hirschsprung's disease and stretched primary mouse colonic smooth muscle cells (SMCs). Furthermore, we demonstrated that persistent stretch-induced Ca2+ overload was originated from extracellular Ca2+ influx and endoplasmic reticulum (ER) Ca2+ release identified by treating with different Ca2+ channel blockers, and was responsible for the persistent activation of MAPK signaling and SCF reduction in colonic SMCs. Our results suggested that Ca2+ overload caused by smooth muscle stretch led to persistent activation of MAPK signaling which might contribute to the decrease of SCF and development of the GMDs.

miRNAs are recently found playing important roles in osteogenesis. In this study, we identified that miR-222-3p decreased during osteogenic differentiation of human mesenchymal stem cells (hBMSCs) using Quantitative Real-Time Reverse Transcription PCR (qRT-PCR). Furthermore, we investigated the effect of miR-222-3p on osteogenic differentiation of hBMSCs. Inhibition of miR-222-3p function in hBMSCs using infection of lentiviruses carrying miR-222-3p specific inhibitor promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Whereas, overexpression of miR-222-3p inhibited osteoblast differentiation of hBMSCs in vitro. Moreover, Smad5 and RUNX2, which are the critical transcription factors in osteogenic differentiation, were predicted to be targets of miR-222-3p by bioinformatic analysis. Overexpression of miR-222-3p in hBMSCs significantly suppressed the protein levels of Smad5 and RUNX2, while inhibition of miR-222-3p increased their protein levels. Furthermore, inhibition of miR-222-3p increased phosphorylation of Smad1/5/8, which regulated the expression of osteogenic genes. Our findings suggest that suppression of miR-222-3p activity promoted osteogenic differentiation hBMSCs through regulating Smad5-RUNX2 signaling axis.

Cervical cancer is one of the most lethal types of cancer in female. Aberrant activation of Wnt/β-catenin signaling pathway has been found to be involved in cervical cancer development and progression, whereas the underlying molecular mechanisms remain poorly understood. The present study showed that NHERF1 was a novel gene associated with both cell proliferation and Wnt signaling pathway in cervical cancer by analysis of differential gene expression and gene cluster for the cervical cancer specimens from GEO data sets. It was further demonstrated in cellular study that NHERF1 inhibition of cervical cancer cell proliferation through Wnt/β-catenin signaling was dependent on α-actinin-4 (ACTN4) expression. A negative association between NHERF1 expression and levels of ACTN4 and β-catenin was found in mouse xenograft model and cervical cancer specimens. Low levels of NHERF1 in cervical cancer specimens were found to associate with activation of cell proliferation and Wnt/β-catenin signaling by gene set enrichment analysis, and also were an independent predictive factor for worse prognosis of cervical cancer patients by Cox regression analysis. These findings demonstrate that NHERF1 inhibits Wnt signaling-mediated proliferation of cervical cancer via suppression of ACTN4, and NHERF1 downregulation may contribute to the progression of cervical cancer. These findings may also shed some lights for understanding the underlying mechanisms of cisplatin resistance and worse prognosis of HPV-inactive cervical cancer patients.

Mucinous colorectal adenocarcinoma (MCA) is characterized by a great mount of extracellular mucus fundamentally composed of Mucin2 (MUC2) which is significantly correlated with the high malignancy and strong invasive ability of MCA. However, rare is known about the underlying mechanism of the mucus accumulation in MCA. Our latest study demonstrated that SCF/c-KIT signaling was highly activated in MCA patients and mouse model, which up-regulated MUC2 transcription. In the present study, we paid a special interest in whether and how SCF/c-KIT signaling promoted mucus secretion by using wild-type (WT) C57BL mice and their littermates who harbor mutational c-kit gene (Wadsm/m), clinical colorectal cancer (CRC) samples, as well as human CRC cell lines. Our results clearly showed that the inner mucus layer of colon was thinner and the intracellular mucin residual was more in Wadsm/m mice than those in WT mice by Alcian blue and PAS staining, suggesting that the mucus secretion process was crippled when SCF/c-KIT signaling was hypo-activated. Inhibiting SCF/c-KIT signaling by Imatinib also resulted in weakened mucus secretion in WT mice. Intraperitoneal administration of MANS which competitively inhibits the activity of the vesicular transport protein MARCKS efficiently reduced mucus secretion in colonic goblet cells of WT mice. Significantly, phosphorylated MARCKS (p-MARCKS) was overtly decreased in colonic mucosa of Wadsm/m mice compared with WT mice, indicating that SCF/c-KIT signaling-regulated mucus secretion was probably mediated by MARCKS activation. Similar results were obtained in MCA patients and mouse model. Moreover, SCF/c-KIT signaling was activated or inhibited in HT-29 and LS174T CRC cells, which potently increased or decreased MARCKS activity, respectively. Finally, we found that PKCδ, a known kinase for MARCKS, was activated in WT and MCA mice along with MARCKS. Inhibition or activation of SCF/c-KIT signaling resulted in decreased or increased PKCδ activity respectively in vitro. In conclusion, we demonstrated that SCF/c-KIT signaling can promote the mucus secretion by activating PKCδ-MARCKS, which provided a new insight into understanding the mechanism of mucus secretion of goblet cells and MCA development.

// Jun Tan 1, 2 , Shu Yang 1, 2, 3 , Ping Shen 1, 3 , Haimei Sun 1, 2, 3 , Jie Xiao 1, 3 , Yaxi Wang 1, 2 , Bo Wu 1, 2, 3 , Fengqing Ji 1, 2, 3 , Jihong Yan 1 , Hong Xue 1 , Deshan Zhou 1, 2, 3 1 Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, P. R. China 2 Beijing Key Laboratory of Cancer Invasion and Metastasis Research, Beijing 100069, P. R. China 3 Cancer Institute of Capital Medical University, Beijing 100069, P. R. China Correspondence to: Deshan Zhou, e-mail: zhoudeshan2008@163.com Keywords: colorectal mucinous adenocarcinoma, C-kit, ETV4, murine model Received: March 24, 2015 Accepted: August 20, 2015 Published: September 02, 2015 ABSTRACT It was reported that the receptor tyrosine kinase (RTK) family often highly expressed in several mucinous carcinomas. In the present study, we established a murine model of colorectal mucinous adenocardinoma (CRMAC) by treating C57 mice [both wild type (WT) and loss-of-function c-kit mutant type (Wads −/− )] with AOM+DSS for 37 weeks and found that c-kit, a member of RTK family, clearly enhanced the tumor cell proliferation by decreasing p53 and increasing cyclin D1 through AKT pathway. Significantly, c-kit strongly promoted tumor cell invasiveness by increasing ETV4, which induced MMP7 expression and epithelial-mesenchymal transition (EMT) via ERK pathway. In vitro up- or down-regulating c-kit activation in human colorectal cancer HCT-116 cells further consolidated these results. In conclusion, our data suggested that the c-kit signaling obviously promoted proliferation and invasion of CRMAC. Therefore, targeting the c-kit signaling and its downstream molecules might provide the potential strategies for treatment of patients suffering from CRMAC in the future.

Diabetic gastroparesis is a common complication of diabetes mellitus (DM) that is characterized by decreased serum insulin and insulin-like growth factor-1 (IGF-1). Despite the fact that insulin treatment not glycemic control potently accelerated gastric emptying in type 1 DM patients, the role of insulin/InsR and IGF-1/IGF-1R signaling in diabetic gastroparesis remains incompletely elucidated. In the present study, type 1 DM mice were established and treated with insulin or Voglibose for 8 weeks. The gastric emptying was delayed from DM week 4 when the gastric InsR and IGF-1R were declined. Meanwhile, the gastric choline acetyltransferase (ChAT) was significantly reduced and the myenteric cholinergic neurones and their fibers were significantly diminished. The production of stem cell factor (SCF) was dramatically repressed in the gastric smooth muscles in DM week 6. TWereafter, interstitial cells of Cajal (ICC) were clearly lost and their networks were impaired in DM week 8. Significantly, compared with Voglibose, an 8-week treatment with insulin more efficiently delayed diabetic gastroparesis development by protecting the myenteric cholinergic neurones and ICC. In conclusion, diabetic gastroparesis was an aggressive process due to the successive damages of myenteric cholinergic neurones and ICC by impairing the insulin/InsR and IGF-1/IGF-1R signaling. Insulin therapy in the early stage may delay diabetic gastroparesis.

Extracellular toxins released by marine toxigenic algae into the marine environment have attracted increasing attention in recent years. In this study, profiling, characterization and quantification of extracellular toxin compounds associated with diarrhetic shellfish poison (DSP) in the culture medium of toxin-producing dinoflagellates were performed using high-performance liquid chromatography–high-resolution mass spectrometry/tandem mass spectrometry for the first time. Results showed that solid-phase extraction can effectively enrich and clean the DSP compounds in the culture medium of Prorocentrum lima (P. lima), and the proposed method achieved satisfactory recoveries (94.80%–100.58%) and repeatability (relative standard deviation ≤9.27%). Commercial software associated with the accurate mass information of known DSP toxins and their derivatives was used to screen and identify DSP compounds. Nine extracellular DSP compounds were identified, of which seven toxins (including OA-D7b, OA-D9b, OA-D10a/b, and so on) were found in the culture medium of P. lima for the first time. The results of quantitative analysis showed that the contents of extracellular DSP compounds in P. lima culture medium were relatively high, and the types and contents of intracellular and extracellular toxins apparently varied in the different growth stages of P. lima. The concentrations of extracellular okadaic acid and dinophysistoxin-1 were within 19.9–34.0 and 15.2–27.9 μg/L, respectively. The total concentration of the DSP compounds was within the range of 57.70–79.63 μg/L. The results showed that the proposed method is an effective tool for profiling the extracellular DSP compounds in the culture medium of marine toxigenic algae.

Background/aim Microsatellite instability (MSI) is a hallmark of genomic instability. In gastric cancer (GC), MSI phenotype is an important molecular subgroup that is often closely correlated with elevated mutation rates on the whole genome level. However, on a single gene level, it is still unknown whether the MSI status of a gene is correlated with the mutational profile of the gene itself. Materials and methods We analyzed intron MSI status and exon mutational profile of TP53 through short tandem repeat (STR) scanning and direct sequencing respectively in gastric cancers and their matched normal tissues. Results MSI status of the TP53 intron was significantly associated with the mutational profile of seven TP53 exon positions (p=0.0416), male patients (p=0.0095), and drinking (p=0.0474), and showed a mild correlation with longer survival time (p=0.0584) and increasing age (p=0.0611). Conclusion TP53 exons tended to mutation in the status of TP53 intron MSI.

Background/Aim: Overexpression of erythropoietin-producing hepatocellular A1 (EPHA1), a member of the EPH super family, is frequently observed in various cancer types. The dysregulated interaction of EPHA1 with its ligand Ephrin A1 has been linked to the progression of ovarian cancer (OC). However, the contribution of EPHA1 in the regulation of the aggressive properties of OC cells remains unknown. Materials and Methods: In this study we investigated the differential expression of EPHA1 in human OC cells. The EPHA1 gene was knocked-down using the CRISPR/Cas9 technique to evaluate its effect on the progressive properties of OC cells. Results: After EPHA1 was knocked-down using a CRISPR/CAS9 genomic editing system in OC cells (SKOV3 and COV504), we observed cell-cycle arrest at the G0/G1 phases in both OC cell lines. Knockdown of EPHA1 in the two OC cells inhibited their aggressive traits, including proliferation, invasion and migration, as well as improving their attachment to extracellular matrix. EPHA1 may play a role in OC through its regulation of multiple signaling pathways, such as matrix metalloproteinase-2 (MMP2), extracellular signal-regulated kinase 2 (ERK2) and proto-oncogene c-MYC. Conclusion: EPHA1 may promote the aggression of some OC cells and, thus, be considered a potential therapeutic target for the treatment of malignant OC.

Overexpression of erythropoietin-producing hepatocellular A1 (EPHA1), a member of the EPH super family, is frequently observed in various cancer types. The dysregulated interaction of EPHA1 with its ligand Ephrin A1 has been linked to the progression of ovarian cancer (OC). However, the contribution of EPHA1 in the regulation of the aggressive properties of OC cells remains unknown.In this study we investigated the differential expression of EPHA1 in human OC cells. The EPHA1 gene was knocked-down using the CRISPR/Cas9 technique to evaluate its effect on the progressive properties of OC cells.After EPHA1 was knocked-down using a CRISPR/CAS9 genomic editing system in OC cells (SKOV3 and COV504), we observed cell-cycle arrest at the GEPHA1 may promote the aggression of some OC cells and, thus, be considered a potential therapeutic target for the treatment of malignant OC.

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

MiR-34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR-34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR-34c and KITLG mRNA expression levels in CRC cell lines, including HT-29, HCT-116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR-34c in the 3' untranslated region (3'UTR) of KITLG mRNA. A dual-luciferase reporter assay further confirmed that KITLG is a direct target of miR-34c. Then, the cell lines were infected with lentiviruses expressing miR-34c or a miR-34c specific inhibitor. Restoration of miR-34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR-34c increased the expression of KITLG protein. The miR-34c-mediated down-regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down-regulation for the tumour-suppressive effects of miR-34c in CRC cells. In conclusion, our results demonstrated that miR-34c might interfere with KITLG-related CRC and could be a novel molecular target for CRC patients.

Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD. © 2013 Wiley Periodicals, Inc.

Hypermethylation often leads to gene silencing; however, the mechanism responsible for the low expression resulting from hypermethylation of the tumor suppressor gene Kelch-like ECH-associating protein 1 (Keap1) in human lung cancer cell lines remains unclear. In this study, using promoter deletion and site mutagenesis assays, we determined that one transcription factor stimulating protein-1 (Sp1) regulatory element in the Keap1 promoter region was important for the transcription of Keap1 in A549 cells. We demonstrated that the transcription factor Sp1 can directly bind to this element in the normal bronchial epithelial BEAS-2B cell line but not in A549 cells, as assessed with chromatin immunoprecipitation (ChIP). EMSAs and supershift assays also showed that CpG island methylation could abrogate Sp1 binding to the Keap1 promoter. Moreover, Keap1 mRNA decreased by 50% after the knock-down of Sp1 with siRNA in BEAS-2B cells, whereas the over-expression of Sp1 led to a dramatic increase in Keap1 promoter activity. The treatment of A549 cells with 5-aza-2'-deoxycytidine restored the binding of Sp1 to the promoter and Keap1 expression. Our results indicate that Sp1 is essential for Keap1 expression and that promoter methylation blocks Sp1 binding in A549 cells. These results demonstrate that hypermethylation may act as an epigenetic gene silencing mechanism, i.e., the inhibition of Sp1 binding to the hypermethylated Keap1 promoter in lung cancer cells, which suggests new approaches to lung cancer treatment.

Erythropoietin-producing hepatocellular A1 (EPHA1) is the first member of the EPH superfamily. Its abnormal expression has been reported in various cancer types. However, the contribution of EPHA1 to the regulation of colorectal cancer cell behaviour remains unknown.In this study, we investigated the expression profile of EPHA1 in human colorectal cancer and its effect on the adhesion and motility of colorectal cancer cells. We used human colorectal cancer specimens and the colorectal adenocarcinoma cell line HRT18 for this purpose.Our cohort screening data showed that in patients with colorectal cancer, low expression of EPHA1 gene is correlated with a remarkably reduced survival. After EPHA1 is knocked-down in colorectal cancer cells using a clustered regularly interspaced short palindromic repeats-associated nuclease 9 (CRISPR-CAS9) genomic editing system, we observed an increase in the spreading and adhesion of HRT18 cells. Moreover, protein array data indicated that the extracellular-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) signaling pathways were activated as a consequence. Inhibition of ERK and JNK proteins with specific inhibitors led to suppression of migration of the colorectal cancer cells.EPHA1 suppresses spreading and adhesion of HRT18 colorectal cancer cells through deactivation of ERK and JNK signaling pathways.

E2F1 promoted miR-34c transcription which reduced its target stem cell factor (SCF) and inhibited colorectal cancer (CRC) cell proliferation. While, SCF increased E2F1 production, suggesting an existence of E2F1-miR-34c-SCF negative feedback loop, which was interrupted by hyper-methylation of miR-34c promoter in CRC cells., Tumour suppressor miR-34c deficiency resulted from hyper-methylation in its promoter is believed to be one of the main causes of colorectal cancer (CRC). Till date, miR-34c has been validated as a direct target of p53; but previous evidence suggested other transcription factor(s) must be involved in miR-34c transcription. In the present study, we in the first place identified a core promoter region (−1118 to −883 bp) of pre-miR-34c which was embedded within a hyper-methylated CpG island. Secondly, E2F1 promoted miR-34c transcription by physical interaction with the miR-34c promoter at site −897 to −889 bp. The transcriptional activating effect of E2F1 on miR-34c was in a p53 independent manner but profoundly promoted in the presence of p53 with exposure to 5-aza-2′-deoxycytidine (DAC). Thirdly, stem cell factor (SCF), a miR-34c target, was specifically reduced upon an introduction of E2F1 which lead to suppression of CRC cell proliferation. The E2F1-suppressed cell proliferation was partially abrogated by additional miR-34c inhibitor, indicating that the anti-proliferation effect of E2F1 was probably through activating miR-34c-SCF axis. Finally, SCF/KIT signalling increased E2F1 production by reducing its proteosomal degradation dependent on PI3K/Akt-GSK3β pathway. In conclusion, our results suggested the existence of E2F1-miR-34c-SCF negative feedback loop which was interrupted by the hyper-methylation of miR-34c promoter in CRC cells and increased cell proliferation.

Normal aging is commonly associated with decreased cognitive functions, which could be conspicuously alleviated by enriched environment (EE) with physical, social, and sensory stimuli, suggesting that aging brain still has intriguing plasticity. Multiple researches have been carried out to explore the structural and the molecular changes in aging brain, which would be considered for evidences that EE regulated brain plasticity. Because there is no significant neuron loss in aging cerebral cortex and the white matter is crucial for cognitive functions, this review focused on the age-related white matter changes and the effects of EE on aged white matter. Data from our stereology laboratory revealed that age-related spatial memory declines had more to do with white matter alterations, which were due to marked demyelination and loss of oligodendrocytes in the white matter. We also demonstrated that EE recovered spatial memory impairment and increased white matter volume by promoting marked remyelination in aged brain. This review approached the issue that EE might contribute to normal aging and be beneficial for those suffering from demyelinated diseases.

The anticancer effects of artesunate (ART) have been well documented. However, its potential against skin cancer has not been explored yet. Herein we reported that 60 μmol/l ART effectively inhibited A431 (human epidermoid carcinoma cells) growth but not that of HaCaT (normal human keratinocyte cells). Our results revealed that ART induced cell cycle arrest at G0/G1 phase through the downregulation of cyclin A1, cyclin B, cyclin D1, Cdk2, Cdk4, and Cdk6. This correlated with the upregulation of p21 and p27. The 5-bromodeoxyuridine incorporation assay also indicated that ART treatment reduced DNA synthesis in a time-dependent manner. Furthermore, ART induced mitochondrial apoptosis, as evidenced by annexin V/propidium iodide staining and western blot analysis. Interestingly, ART-induced apoptosis diminished under iron-deficient conditions but intensified under iron-overload conditions. Taken together, these findings demonstrated the potential of ART in treating skin cancer through the induction of G0/G1 cell cycle arrest and iron-mediated mitochondrial apoptosis and supported further investigations in other test systems.

Although it is well known that the reduction of interstitial cells of Cajal (ICCs) is associated with several gastrointestinal motility disorders in clinic, it is unknown whether the mature ICCs still have an active plasticity in adult mammals. This study focused on the issues of the reduction of ICCs during Imatinib administration and the recovery of ICCs following drug withdrawal in the small intestine of adult guinea pigs. ICCs were revealed by immunofluorescence on whole mount preparations with anti-Kit, alpha-smooth muscle actin, (alpha-SMA), and 5-bromo-2'-deoxyuridine (BrdU) antibodies. Moreover, the occurrence of apoptosis was also assayed. Imatinib treatment led to a gradual reduction of ICCs in number around the myenteric plexus and deep muscular plexus, which was dependent on the time but no apoptosis of ICCs was detected with the TUNEL method. During Imatinib treatment, some ICC-like cells were double labeled for Kit and alpha-SMA and a few ICC-like cells were only stained with alpha-SMA. When Imatinib was discontinued, the number of ICCs recovered to normal within 32 days. During this time, some proliferating ICCs were demonstrated by double labeling with Kit and BrdU antibodies. Our results indicated that Kit signaling was essential for the maintenance of survival and proliferation of the mature ICCs in the small intestine of adult guinea pigs. Moreover, ICCs might transdifferentiate to a type of alpha-SMA(+) cells, perhaps a phenotype of smooth muscle cells, when there is a loss-of-function of Kit.

This study aimed at evaluating whether apoptosis of interstitial cells of Cajal (ICC), smooth muscle cells (SMC), and enteric neurons was involved in a guinea pig model of intestinal ischemia and reperfusion injury. The small intestinal segments were resected at either 6 (I60/R6h) and 12 h (I60/R12h) or 7 (I60/R7d) to 14 (I60/R14d) days after 60 min intestinal ischemia in the adult guinea pigs and studied by immunohistochemistry with anti-Kit, 5-bromo-2'-deoxyuridine (BrdU), alpha-smooth muscle actin, vimentin, and beta-tublin III antibodies. Also, apoptosis was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. In the I60/R12h injury, there was a approximately 50% decrease of Kit+ cells in cell numbers at the level of myenteric plexus and a number of Kit-/vimentin-positive cells were labeled by TUNEL. Also, a few SMC and enteric neurons were TUNEL positive. The Kit+ ICC recovered to normal and a number of Kit-/BrdU-double-positive cells were observed in the I60/R14d group. Our results indicated that the intestinal I/R injury could lead to apoptosis of ICC, SMC, and enteric neurons which may contribute to the gastrointestinal motility disorders, and proliferation was involved in the recovery of ICC.

This paper aimed at investigating the alterations in interstitial cells of Cajal (ICCs) in the murine small intestine from 0-day to 56-day post-partum (P0-P56) by immunohistochemistry. The Kit+ ICCs, which were situated around myenteric nerve plexus (ICC-MY) formed a loose cellular network at P0 which changed into an intact one before P32. The density of ICC-MY increased from P0 to P12, and then decreased until P32. In contrast, the estimated total amount increased more than 15-fold at P32 than that at P0. Some Kit+/BrdU+ cells were observed at 24 h after one BrdU injection to the different-aged mice, and the number decreased from P2 to P24 and vanished at P32. Actually a few Kit+/BrdU+ cells can be observed at 1 h after one BrdU injection at P10, and the amount doubled at 24 h along with paired Kit+/BrdU+ cells. A number of BrdU+ ICCs were also labeled with CD34, CD44 and insulin-like growth factor I receptor. About 65% ICCs were BrdU+ at P32 after daily BrdU injection from P0. Our results indicate that an age-dependent proliferation is involved in the postnatal development of ICC-MY which increase greatly in cell numbers and proliferative ICCs may originate from ICCs progenitor cells.