Atractylodes macrocephala Koidz promotes intestinal epithelial restitution via the polyamine--voltage-gated K+ channel pathway.

Atractylodes macrocephala Koidz (AMK) has been used widely as a digestive and tonic in traditional Chinese medicine. AMK has shown noteworthy promoting effect on intestinal epithelial cell migration, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via IEC-6 cell migration model. A wounding model of IEC-6 cells was induced by a single-edge razor blade along the diameter of six-well polystyrene plates. The cells were grown in control cultures and in cultures containing spermidine (5 μmol/L, SPD, reference drug), alpha-difluoromethylornithine (2.5 mmol/L, DFMO, polyamine inhibitor), AMK (50, 100, and 200 μg/mL), DFMO plus SPD and DFMO plus AMK for 24h. The membrane potential (MP) and cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) were detected by flow cytometry, and polyamines content was determined via high-performance liquid chromatography (HPLC). The expression of Kv1.1 mRNA and protein levels were assessed by RT-qPCR and Western blot analysis, respectively. Cell migration assay was carried out using the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. (1) Treatment with AMK caused significant increases in cellular polyamines content, membrane hyperpolarization, an elevation of [Ca(2+)]cyt and an acceleration of cell migration in IEC-6 cells, as compared to control group. (2) AMK not only reversed the inhibitory effects of DFMO on the polyamines content, MP, and [Ca(2+)]cyt but also restored IEC-6 cell migration to control levels. (3) The Kv1.1 mRNA and protein expression were significantly increased by AMK treatment in control and polyamine-deficient IEC-6 cells. The results of our current studies revealed that treatment with AMK significantly stimulates the migration of intestinal epithelial cells through polyamine-Kv1.1 channel signaling pathway, which could promote the healing of intestinal injury. These results suggest the potential usefulness of AMK to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.

Song HP ，Li RL ，Chen X ，Wang YY ，Cai JZ ，Liu J ，Chen WW ... -  《-》

Atractylodes macrocephala Koidz stimulates intestinal epithelial cell migration through a polyamine dependent mechanism.

Atractylodes macrocephala Koidz (AMK), a valuable traditional Chinese herbal medicine, has been widely used in clinical practice for treating patients with disorders of the digestive system. AMK has shown noteworthy promoting effect on improving gastrointestinal function and immunity, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via intestinal epithelial (IEC-6) cell migration model. A cell migration model of IEC-6 cells was induced by a single-edge razor blade along the diameter of the cell layers in six-well polystyrene plates. After wounding, the cells were grown in control cultures and in cultures containing spermidine (5μM, SPD, reference drug), alpha-difluoromethylornithine (2.5mM, DFMO, polyamine inhibitor), AMK (50, 100, and 200mg/L), DFMO plus SPD and DFMO plus AMK for 12h. The polyamines content was detected by high-performance liquid chromatography (HPLC) with pre-column derivatization. The Rho mRNAs expression levels were assessed by Q-RT-PCR. The Rho and non-muscle myosin II proteins expression levels were analyzed by Western blot. The formation and distribution of non-muscle myosin II stress fibers were monitored with immunostaining techniques using specific antibodies and observed by confocal microscopy. Cell migration assay was carried out using inverted microscope and the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. (1) Treatment with AMK caused significant increases in cellular polyamines content and Rho mRNAs and proteins expression levels, as compared to control group. Furthermore, AMK exposure increased non-muscle myosin II protein expression levels and formation of non-muscle myosin II stress fibers, and resulted in an acceleration of cell migration in IEC-6 cells. (2) Depletion of cellular polyamines by DFMO resulted in a decrease of cellular polyamines levels, Rho mRNAs and proteins expression, non-muscle myosin II protein formation and distribution, thereby inhibiting IEC-6 cell migration. AMK not only reversed the inhibitory effects of DFMO on the polyamines content, Rho mRNAs and proteins expression, non-muscle myosin II protein formation and distribution, but also restored cell migration to control levels. The results obtained from this study revealed that AMK significantly stimulates the migration of IEC-6 cells through a polyamine dependent mechanism, which could accelerate the healing of intestinal injury. These findings suggest the potential value of AMK in curing intestinal diseases characterized by injury and ineffective repair of the intestinal mucosa in clinical practice.

Song HP ，Li RL ，Zhou C ，Cai X ，Huang HY ... -  《-》

Polysaccharide extracts of Astragalus membranaceus and Atractylodes macrocephala promote intestinal epithelial cell migration by activating the polyamine-mediated K(+) channel.

Astragalus membranaceus (Radix Astragali, RA) and Atractylodes macrocephala (Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control media or media containing spermidine (5 μmol·L-1, SPD), alpha-difluoromethylornithine (2.5 mmol·L-1, DFMO), 4-Aminopyridine (40 μmol·L-1, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L-1), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca2+ ([Ca2+]cyt) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca2+]cyt and accelerated migration of IEC-6 cells, compared with the controls (P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca2+]cyt, but also restored IEC-6 cell migration to control level (P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased (P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0 : 14.1 : 0.3 : 19.9 : 181.3 : 6.3 in RA and 1.0 : 4.3 : 0.1 : 5.7 : 2.8 : 2.2 in RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.

Zeng D ，Hu C ，Li RL ，Lin CQ ，Cai JZ ，Wu TT ，Sui JJ ，Lu WB ，Chen WW ... -  《-》

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 ... -  《-》

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration.

Wang JY ，Wang J ，Golovina VA ，Li L ，Platoshyn O ，Yuan JX ... -  《AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY》