Healing Effect Of Rheum Tanguticum Polysaccharides (RTP) On Intestinal Epithelial Cell Injury And Its Possible Mechanism

BACKGROUNDRheum Tanguticum Maxim., a traditional Chinese medicine, is usually used for the treatment of gastrointestinal diseases, and demonstrated its . remarkable curative effects. Pharmaceutical chemistry and pharmacology studies have showed that polysaccharide (RTP) is one of its major active ingredients. The water soluble polysaccharides, with 5 different molecular weight {Rheum tanguticum polysaccharides, RTP₁-5) were isolated from Rheum Tanguticum Maxim in our lab. Animal experiment displayed that RTP₁ (molecular weight 6⁸×105) could prevent TNBS-induced colits in rat. And this effect was developed via decreasing the ulcer area and hyperaemia, as well as reducing the mucosal injury induced by peroxide oxidants. It also demonstrated modulating effects on local imunne system. However, up to now, it remains unclear whether the repairing effect of RTP₁ on mucosa was related to its direct protective action and what its possiblemechanism is.AIMThe present study was therefore designed to investigate the healing effects of RTPi on intestinal epithelial cell injury and its links. We also sought to determine the underlying mechanisms of these effects so that we may provide theory basis for Rheum Tanguticum Maxim, and its polysaccharids on the treatment of gastrointestinal pathological changes.METHODSIEC-6 cell, a cell line derived from normal rat intestinal epithelium was used in our experiment. In RTPi groups, cells were pretreated with RTPi for 24h for further studies.1. Effects of RTPi on IEC-6 cell proliferation, restitution, differentiation and its possible mechanism.1.1 Proliferation: Cells were divided into 6 groups and incubated with different concentrations of RTPj (0,3,10,30,100,300ug/ml) for 24h. MTT assay was introduced to assess the effect of RTPi on cell proliferation.1.2 Restitution: In vitro wounding model was used to detect the effects of RTPi on cell restitution. Cells were divided into 2 groups (Control and RTPilOOug/ml-treated group) and cultured until confluent. The changes in the cell free area over time were monitored under phase-contrast microscope and were quantitatively by analyzing the size of the cell-free area.1.3 Differentiation: H&E staining was used to survey cell differentiation as morphological criteria. Alkaline phosphatase activity in IEC-6 cells wasdetected by spectrophotometry using an assay kit.1.4 Effect of DFMO on RTPi-induced IEC-6 cell proliferation: IEC-6 cells were co-treated with RTPi and DFMO: Cells were divided into 4 groups: (a)Control, (b) RTPilOO|ig/ml, (c) RTPilOOug/ml +5mM DFMO,(d) RTPilOOug/ml +10mM DFMO. After different treatment, MTT assay was used to assess if there exist any changes when co-treated with ODC antagonist DFMO.1.5 Putrescine concentration in IEC-6 cells: Cells were incubated with RTPi (lOug/ml, 30ug/ml and lOOug/ml) or co-treated with RTPi and DFMO, putrescine concentration was assessed by spectrophotometry.1.6 Western blot analysis was used to detect ODC and c-Myc protein expression in cells treated with different concentrations of RTPi.2. Cytoprotective effect of RTPi against oxidative stress-induced intestinal cell injury and its possible mechanismHydrogen peroxide (t^Oa, lOOumol/L, incubation for 2h) was selected to induce cell injury. Cells were divided into control group, H2O2 group and RTP1+H2O2 group. Following methods were adopted:2.1 Effects of RTPi on IEC-6 cell viability: Effects of RTPi on IEC-6 cell morphological changes were monitored by phase contrast microscopy. And MTT assay was employed to detect cell viability. LDH activity in the supernatant was measured by spectrophotometry.2.2 Effects of RTPi on H2O2-induced IEC-6 cell injury: GSH-Px and SOD activity as well as the production of MDA were detected by spectrophotometry assay using assay kits.2.3 Effects of RTPi on IEC-6 cell apoptosis: Acridine orange staining was used as morphological observation, while flowcytometry analysis using Annexin V/PI staining was adopted to measure the apoptotic ratio of IEC-6 cells.2.4 Caspase 3 activation was detected by Western blot analysis.2.5 Immunochemical staining was used to detect Bcl-2 and Bax protein expression in IEC-6 cells.2.6 Production of TNF-a in the supernatant was measured by ELISA.RESULTS1. RTPi could dose-dependently enhance restitution, proliferation and differentiation of IEC-6 cells. There existed significant difference by comparing RTPi-treated groups with control group (P<0.05 or P<0.01). It also significantly promoted putrescine concentration in IEC-6 cells in a concentration-dependent manner. ODC and c-Myc protein expression was also increased as a result of this process. DL-a-difluoromethyl-ornithine (DFMO) repressed the cell proliferation and putrescine concentration induced byRTP,.2. The pretreatment of RTPi on IEC-6 cells demonstrated strong protective effect against EbCVinduced insult. Following exposure to H2O2, cell number decreased markedly. Most cells demonstrated round shape and lost adhesion. Pretreatment with RTPi could alter morphological appearance of IEC-6 cells. Treatment of the cells with RTPi at lOug/ml lOOug/ml prior to H2O2 exposure significantly elevated the survival of IEC-6 cells and decreased LDH activity. In parallel, the H2O2-induced production of MDA in culturedcells was markedly reduced accompanied by the enhanced GSH-Px and SOD activity in RTPi-treated cells in a dose-dependent fashion. RTPi pretreatment significantly attenuated H2O2 induced apoptosis in IEC-6 cells. Compared with H2O2 treated IEC-6 cells (the apoptotic ratio is 31.3%), RTPi30u-g/ml and lOOug/ml pretreated cells showed less apoptotic ratio (24.4% and 21.5%, respectively).Furthermore, it was found that RTPi pretreatment could inhibit H2O2-induced activation of caspase 3. Immunochemical staining showed that the expression of anti-apoptotic protein Bcl-2 was increased accompanied with suppressed Bax protein expression. Again, this effect was supportted by the measurement of TNF-a production by ELISA. In RTPi pretreated groups, the production were much lower than that of H2O2 group (PO.Ol or P<0.05 )CONCLUSION1. RTPi could accelerate mucosal healing by enhancing cell proliferation, migration and differentiation, three critical steps of intestinal mucosal healing, while ODC and c-Myc are closely associated with this effect. It was considered that the stimulating proliferation effect of RTPi might be through the upregulation of c-myc and c-fos, which subsequently activated ODC expression.2. RTPi pretreatment could lower the risk of oxidative stress-induced cellular damage and programmed cell death by increasing the activity of GSH-Px and SOD and decreasing the MDA content and LDH activity. RTPi decreased the susceptibility of cells to oxidative stress-induced cellular injury and cell...