Anti-inflammatory Effects Of Oryzanol In Colitis Induced By Dextran Sulphate Sodium (DSS) In Mice And Its Mechanism Study

Oryzanol, a biological substance in rice bran, is a complex of cycloartenol and phytosterol alcohol ferulic acid esters. It has many physiological functions, such as scavenging free radicals, antioxidation, reducing blood lipid, reducing blood glucose, anticancer and so on. Recent studies have been found oryzanol possibly exists the function of anti-inflammation. However, the anti-inflammatory mechanism was still in the stage of preliminary study. Ulcerative colitis (UC) is idiopathic, chronic and relapsing inflammatory disorders of the gastrointestinal tract. Extentive research have been revealed cytokines play a crucial role in modulating inflammation which is regulated by a delicate balance of pro-inflammatory and anti-inflammatory cytokines. However, its aetiology remains unclear. In this paper, dextran sodium sulphate (DSS)-induced ulcerative colitis in C57BL/6mice in vivo and lipopolysaccharide (LPS)-stimulated inflammatory model in murine RAW264.7macrophages in vitro were established to evaluate the anti-inflammatory activity and study its mechanism of oryzanol. The paper provided theoretical and experimental basis for further research.1The anti-inflammatory effect of oryzanol in colitis induced by DSS in C57BL/6miceIn this study, a mouse model of1.0%DSS-induced inflammatory was used to investigate the effects of oryzanol on ulcerative colitis12days after DSS administration. The mice were randomly divided into three groups (n=20):Group A (the normal group); Group B (the experimental colitis group with administration of1.0%DSS solution for12days); Group C (oryzanol treatment group with administration of1.0%DSS solution and injection of150mg/kg oryzanol). All mice were sacrificed after14days.The results:(1) DAI score revealed that DSS induced a severe mucosal colitis, with a remarkably decrease in body weight, increase in thickness of the muscle layer, increase in weight and decrease in length of the colitis tissues, increase in weight of spleen, intestinal bleeding and diarrhoea. (2) The activity of MPO:the activity of MPO of A, B and C groups are0.2950±0.0536,0.8260±0.2031and0.5752±0.1919U/g, respectively. The activity of MPO of group B was significantly higher than those of the group A and group C (both P<0.01).Comparing with group B, group C reduced the activity of MPO (P<0.05).(3) The relative expression level of TNF-a and IL-6mRNA:The expression level of IL-6mRNA are1.36±0.11,51.11±13.73and40.75±13.81, while TNF-a mRNA are1.55±0.23,13.11±4.07and8.12±1.64, respectively. Expression level of pro-inflammatory cytokines mRNA were significantly increased in DSS-induced colitis tissues. Oryzanol (150mgkg-1d-1p.o.) markedly inhibited these inflammatory reactions.In vivo assay demonstrated that oryzanol which has anti-inflammatory effect has protection on DSS-induced colitis and immune system disorders.2The anti-inflammatory effect of oryzanol in LPS-stimulated RAW264.7macrophagesFurther study was researched in vitro anti-inflammatory of oryzanol.(1μg/ml) LPS-stimulated murine RAW264.7macrophages have been established to explore the anti-inflammatory molecular mechanism of oryzanol (6.25、12.5、25μmol/mL). The culture medium was collected and NO production was determined by nitrate reductive enzymatic method. ELISA was used to study the effects of oryzanol on secretion of TNF-a and IL-6. Real time PCR method was adopted to research the effects of oryzanol on expression level of iNOS, TNF-a and IL-6mRNA in LPS-induced RAW264.7cells.The results:(1) Effects of oryzanol on TNF-a and IL-6secretion in LPS-induced RAW264.7cells.①TNF-α:After treatment with1μg/ml LPS for24h, compared with control group, TNF-a secretion was significantly increased from112.29±12.33to674.56±101.57ng/L (P<0.01). After treatment with1μg/ml LPS and oryzanol6.25～25μmol/L for24h, TNF-a secretion were639.40±74.52,477.94±31.88,271.44±79.47ng/L in oryzanol groups, respectively. Compared with LPS group, oryzanol decreased LPS-induced TNF-a production (P<0.05,P<0.01).②IL-6:After treatment with1μg/ml LPS for24h, compared with control group, IL-6secretion was significantly increased from11.03±0.17to89.26±3.73pg/mL (P<0.01). After treatment with1μg/ml LPS and oryzanol6.25～25μmol/L for24h, IL-6secretion amount were84.93±2.60,80.84±1.20,61.43±2.70pg/mL in oryzanol groups, respectively. Compared with LPS group,12.5and25μmol/L oryzanol significantly decreased LPS-induced IL-6production (P<0.05, P<0.01). No obvious effect was found in6.25μmol/L oryzanol group (P>0.05). Oryzanol inhibited TNF-a and IL-6secretion level in a dose-dependent manner, consistent with cytokine mRNA expression.(2) Oryzanol also inhibited NO synthesis in LPS-stimulated murine RAW264.7macrophages by inhibiting the expression of iNOS mRNA.In vitro assay demonstrated that Oryzanol has positive effect on inflammation in LPS-stimulated RAW264.7macrophages.