Polysaccharides from Panax ginseng promote intestinal epithelial cell migration through affecting the Ca(2+) related regulators.

Panax ginseng, a key herbal medicine of replenishing Qi and tonifying Spleen, is widely used in the treatment of gastrointestinal diseases in East Asia. In this study, we aim to investigate the potential effects and mechanisms of polysaccharides from P. ginseng (PGP) on intestinal mucosal restitution which is one of the crucial repair modalities during the recovery of mucosal injury controlled by the Ca2+ signaling. Rat model of intestinal mucosal injury was induced by indomethacin. The fractional cell migration was carried out by immunohistochemistry staining with BrdU. The morphological observations on intestinal mucosal injury were also performed. Intestinal epithelial cell (IEC-6) migration in vitro was conducted by scratch method. Western-blot was adopted to determine the expressions of PLC-γ1, Rac1, TRPC1, RhoA and Cav-1. Immunoprecipitation was used to evaluate the levels of Rac1/PLC-γ1, RhoA/TRPC1 and Cav-1/TRPC1. The results showed that PGP effectively reduced the assessment of intestinal mucosal injury, reversed the inhibition of epithelial cell migration induced by Indomethacin, and increased the level of Ca2+ in intestinal mucosa in vivo. Moreover, PGP dramatically promoted IEC-6 cell migration, the expression of Ca2+ regulators (PLC-γ1, Rac1, TRPC1, Cav-1 and RhoA) as well as protein complexes (Rac1/PLC-γ1, Cav-1/TRPC1 and RhoA/TRPC1) in vitro. PGP increases the Ca2+ content in intestinal mucosa partly through controlling the regulators of Ca2+ mobilization, subsequently promotes intestinal epithelial cell migration, and then prevents intestinal mucosal injury induced by indomethacin.

Zhu H ，Cao J ，Liang X ，Luo M ，Wang A ，Hu L ，Li R ... -  《-》

Atractylenolide I stimulates intestinal epithelial repair through polyamine-mediated Ca(2+) signaling pathway.

An impairment of the integrity of the mucosal epithelial barrier can be observed in the course of various gastrointestinal diseases. The migration and proliferation of the intestinal epithelial (IEC-6) cells are essential repair modalities to the healing of mucosal ulcers and wounds. Atractylenolide I (AT-I), one of the major bioactive components in the rhizome of Atractylodes macrocephala Koidz. (AMR), possesses multiple pharmacological activities. This study was designed to investigate the therapeutic effects and the underlying molecular mechanisms of AT-I on gastrointestinal mucosal injury. Scratch method with a gel-loading microtip was used to detect IEC-6 cell migration. The real-time cell analyzer (RTCA) system was adopted to evaluate IEC-6 cell proliferation. Intracellular polyamines content was determined using high performance liquid chromatography (HPLC). Flow cytometry was used to measure cytosolic free Ca2+ concentration ([Ca2+]c). mRNA and protein expression of TRPC1 and PLC-γ1 were determined by real-time PCR and Western blotting assay respectively. Treatment of IEC-6 cells with AT-I promoted cell migration and proliferation, increased polyamines content, raised cytosolic free Ca2+ concentration ([Ca2+]c), and enhanced TRPC1 and PLC-γ1 mRNA and protein expression. Depletion of cellular polyamines by DL-a-difluoromethylornithine (DFMO, an inhibitor of polyamine synthesis) suppressed cell migration and proliferation, decreased polyamines content, and reduced [Ca2+]c, which was paralleled by a decrease in TRPC1 and PLC-γ1 mRNA and protein expression in IEC-6 cells. AT-I reversed the effects of DFMO on polyamines content, [Ca2+]c, TRPC1 and PLC-γ1 mRNA and protein expression, and restored IEC-6 cell migration and proliferation to near normal levels. Our data demonstrate that AT-I stimulates intestinal epithelial cell migration and proliferation via the polyamine-mediated Ca2+ signaling pathway. Therefore, AT-I may have the potential to be further developed as a promising therapeutic agent to treat diseases associated with gastrointestinal mucosal injury, such as inflammatory bowel disease and peptic ulcer.

Song HP ，Hou XQ ，Li RY ，Yu R ，Li X ，Zhou SN ，Huang HY ，Cai X ，Zhou C ... -  《-》

Effect of polysaccharides from Sijunzi decoction on Ca(2+) related regulators during intestinal mucosal restitution.

Sijunzi decoction, a representative Chinese herbal formula of reinforcing Qi strengthening spleen, has been widely used for treating patients with gastrointestinal diseases and spleen-deficiency syndrome, however, the exact effects and mechanisms are remained unclear. The migration of intestinal epithelial (IEC-6) cells has been demonstrated to be one of the major repair modalities during the healing of mucosal wounds. Ca2+ is the key factor in regulating cell migration. Thus, this study aimed to elucidate the potential effects and mechanisms of polysaccharides (SJZDP) extracted from Sijunzi decoction in intestinal mucosal restitution. Cell migration was detected by scratch method with micropipette tip. Western blotting was adopted to evaluate the expression of STIM1 and STIM2 proteins. Immunofluorescence was carried out to assess the translocation of STIM1 protein. Immunoprecipitation was used to determine the levels of STIM1/TRPC1 and STIM1/STIM2 complexes. A indomethacin-induced intestinal mucosal injury rat model was applied. The content of polyamines in intestinal mucosa was detected by high-performance liquid chromatography. The morphological changes in the intestinal mucosa were observed at the end of animal experiment. The results showed that treatment with SJZDP significantly promoted cell migration, enhanced the level of STIM1 protein and STIM1/TRPC1 complexes, reduced the level of STIM2 protein and STIM1/STIM2 complexes. Further more, SJZDP exposure promoted the translocation of STIM1 to the plasma membrane. In vivo experiment results, the administration of SJZDP effectively reduced intestinal mucosal injury. These results revealed that SJZDP promotes intestinal epithelial restitution after wounding, presumably by regulating cellular levels of STIM1 and STIM2 differentially, stimulating the translocation of STIM1, and inducing STIM1/TRPC1 association as well as decreasing the level of STIM1/STIM2.

Shi Y ，Zhu H ，Li R ，Wang D ，Zhu Y ，Hu L ，Chen W ... -  《-》

Rac1 promotes intestinal epithelial restitution by increasing Ca2+ influx through interaction with phospholipase C-(gamma)1 after wounding.

Rao JN ，Liu SV ，Zou T ，Liu L ，Xiao L ，Zhang X ，Bellavance E ，Yuan JX ，Wang JY ... -  《-》

RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding.

Early mucosal restitution occurs as a consequence of epithelial cell migration to resealing of superficial wounds after injury. Our previous studies show that canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca(2+) channel (SOC) in intestinal epithelial cells (IECs) and plays an important role in early epithelial restitution by increasing Ca(2+) influx. Here we further reported that RhoA, a small GTP-binding protein, interacts with and regulates TRPC1, thus enhancing SOC-mediated Ca(2+) entry (SOCE) and epithelial restitution after wounding. RhoA physically associated with TRPC1 and formed the RhoA/TRPC1 complexes, and this interaction increased in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1). Inactivation of RhoA by treating IEC-TRPC1 cells with exoenzyme C3 transferase (C3) or ectopic expression of dominant negative RhoA (DNMRhoA) reduced RhoA/TRPC1 complexes and inhibited Ca(2+) influx after store depletion, which was paralleled by an inhibition of cell migration over the wounded area. In contrast, ectopic expression of wild-type (WT)-RhoA increased the levels of RhoA/TRPC1 complexes, induced Ca(2+) influx through activation of SOCE, and promoted cell migration after wounding. TRPC1 silencing by transfecting stable WT RhoA-transfected cells with siRNA targeting TRPC1 (siTRPC1) reduced SOCE and repressed epithelial restitution. Moreover, ectopic overexpression of WT-RhoA in polyamine-deficient cells rescued the inhibition of Ca(2+) influx and cell migration induced by polyamine depletion. These findings indicate that RhoA interacts with and activates TRPC1 and thus stimulates rapid epithelial restitution after injury by inducing Ca(2+) signaling.

Chung HK ，Rathor N ，Wang SR ，Wang JY ，Rao JN ... -  《-》