AIM: To describe the effect of Rheum tanguticum polysaccharide (RTP) on hydrogen peroxide-induced human intestinal epithelial cell injury.

METHODS: Hydrogen peroxide (100 μmol/L) was introduced to induce human intestinal epithelial cell injury. Cells were pretreated with RTP (30,100,300 μg/mL) for 24 h before exposure to hydrogen peroxide. Cell viability was detected by MTT assay and morphological observation. Acridine orange staining and flow cytometry were performed to assess cell apoptosis. Lactate dehydrogenase (LDH) activity, production of malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured by spectrophotometry with corresponding assay kits.

RESULTS: Following exposure to H₂O₂, a marked decrease in cell survival and SOD activity, increased production of MDA, LDH leakage and cell apoptosis were found. Pretreatment of the cells with RTP could significantly elevate cell survival, SOD activity and decrease the level of MDA, LDH activity and cell apoptosis.

CONCLUSION: RTP may have cytoprotective and anti-oxidant effects against H₂O₂-induced intestinal epithelial cell injury by inhibiting cell apoptosis and necrosis. This might be one of the possible mechanisms of RTP for the treatment of ulcerative colitis in rats.

Intestinal epithelium acts as a gateway that allows absorption of dietary nutrients and restricts uncontrolled entry of luminal antigens. This selective barrier function is critical for maintaining mucosal immune homeostasis, as evidenced in intestinal inflammation associated with loss of epithelial integrity[1].

Epithelial apoptosis, in the gastrointestinal tract, is normally restricted to superficial cells, but in pathological states of inflammation or infection, apoptotic cell death can be far more expansive[2]. Mucosal lesion in inflammatory bowel disease (IBD) is characterized by dense inflammatory cell infiltrate mainly comprised of neutrophils, macrophages and lymphocytes. Although many inflammatory mediators secreted by these cells, including cytokines IL-1, IL-6, IL-8, IL-10, TNF-α and leukotrienes together with luminal bacterial products such as bacterial lipopolysaccharide (LPS) and chemotactic peptide fMLP, have been implicated in the mucosal injury observed in IBD[3], and the molecules that mediate tissue damage remain poorly understood[4-7]. Recent indirect evidence has, however, implicated reactive oxygen and nitrogen species (RONS) such as nitric oxide (NO), superoxide (O₂^-), peroxynitrite (ONOO^-), hydrogen peroxide (H₂O₂), and hypochlorite (OCl^-) in the pathogenesis of mucosal lesion[8-11].

Rheum tanguticum Maxim ex Balf is a well-known traditional Chinese medicine especially for the treatment of gastrointestinal diseases. Polysaccharide is one of its major active ingredients. Our previous studies have demonstrated that polysaccharide extracted from Rheum tanguticum (RTP) can prevent 2, 4, 6-trinetrophene sulphonic acid (TNBS) - induced ulcerative colitis in rats[12]. The present study was mainly conducted to describe whether RTP had protective effects on intestinal epithelial cells against hydrogen peroxide-induced acute oxidant stress in vitro.

Epithelial apoptosis is increased in inflammation or infection[2]. Apoptotic cell death is accompanied with activation of various cell death pathways including caspases, ceramide, altered gene expression, mitochondrial dysfunction and consumption of ATP[4,5].

Although these signals and metabolic events may be important in the regulation of cell death, cell death pathways are also associated with the secondary production of oxidants that appears to contribute to the magnitude of cell death. The intracellular redox level, in particular, has been shown to play a critical role[13].

There is ample evidence that cell death is associated with increased intracellular levels of reactive oxygen species (ROS)[14,15]. This increase in ROS may be due to overproduction of ROS from extracellular or intracellular processes, or there may be a decrease in endogenous antioxidant defense. Much work in recent years has been centered on such intracellular antioxidants as superoxide dismutase, GSH, and catalase. Alterations in activities of these antioxidant systems have been implicated as causes of diseases as well as cell death[16].

Evidence for the involvement of reactive oxygen metabolites is found in inflammatory bowel disease in man and in animal models of intestinal inflammation. Superoxide and hydrogen peroxide generated by activated leukocytes cause damage to the mucosa in intestinal inflammation[15]. Reactive oxygen radicals are unavoidably formed in biochemical processes, and several enzymes are designed to inactivate those metabolites under physiological conditions including cytochrome oxidase, superoxide dismutase and catalase. It has been demonstrated that activated leukocytes form reactive oxygen metabolites and oxidative stress is one of the causes for tissue injury during inflammatory bowel diseases[17].

Polysaccharide is one of the major active ingredients of Rheum tanguticum. Polysaccharide abstracted from Rheum tanguticum can stimulate the function of phagocytes, thus protecting livers function and others[12,18]. Here, polysaccharide with molecular weight 65-80 kDa, has strong anti-oxidant and protective effects against H₂O₂-induced cell injury in cultured HIEC cells.

Pre-treatment with RTP at 300 μg/mL could preserve over 90% of cell survival after H₂O₂ insult and decrease LDH leakage and cell apoptosis. The isolated polysaccharide also inhibits the production of MDA and the activity of SOD caused by exogenous H₂O₂. Thus, RTP may serve as a useful protective agent that mitigates the oxidative stress.

Free radical molecules can lead to damage or destruction of a variety of tissues. Excessive reactive oxygen species result in lipid peroxidation, oxidation of proteins and damage of DNA. Cells are often equipped with several antioxidants for the prevention of free radical damage. SOD and GSH-Px, along with other non-enzymatic antioxidants, such as ascorbate and GSH, serve as a detoxifying system to prevent cell damage caused by reactive oxygen species, and among these, antioxidant enzymes play a pivotal role. The combined action of GSH-Px and SOD provides a repair mechanism for oxidized membrane components. Exposure of cultured cells to free radicals results in an imbalance in energy metabolism in addition to the deleterious effects of hydroxyl and peroxyl radicals on membrane lipids and proteins. Therefore, cellular function is impaired, which finally causes cell death[19].

In conclusion, H₂O₂-induced decrease in cell survival is correlated with reduction in SOD activity, enhancement in cell apoptosis and MDA content. RTP may have protective effects against H₂O₂-induced intestinal epithelial cell injury. Further studies are needed to confirm the exact mechanism of these effects.