Study The Effect Of Octacosanol On Anti-inflammation And Its Molecular Mechanism

N-octacosanol is a kind of monohydric saturated aliphatic alcohol[CH3(CH2)26CH2OH]with many physiological functions,mainly exist in rice bran,sugar cane and many other natural plants in the form of wax ester.In China,the production of rice is rich,and the annual output of processing by-product of rice bran was more than 10 million tons.Therefore,octacosanol,active substances come from rice bran,studying its physiological function and molecular mechanisms have important practical and economic significance.It will promote to developing and utilizing of rice and its by-products.Because of its stable chemical properties,octacosanol is almost non-toxic to humans.Studies have shown that octacosanol has physiological functions such as anti-fatigue,promoting metabolism and improving cardiovascular function.Recent studies indicated that octacosanol may also have anti-inflammatory biological activity,but the molecular mechanism of its anti-inflammatory function is not clear.In this study,the inflammation model of RAW264.7 cells induced by lipopolysaccharide(LPS)and the model of murine colon inflammation induced by sodium dextran sulfate(DSS)were used to investigate the anti-inflammatory effect and molecular mechanism of octacosanol.Effects of octacosanol on macroscopic phenotype and tissue injury in colitis mouse.In this experiment,ICR mice were fed with 3.0%DSS for 11 days to establishenteritis model.The mice in the protection group were fed with 100 mg/kg/day octacosanol suspension for 2 days in advance.The mice in the demage group showed symptoms including weight loss,lazy movement,drowsiness,decreased hair gloss and visible blood in the stool;disease activity index(DAI)continues to rise.The colonic congestion,ulcers,edema and other symptoms lead to thickening of the bowel wall,increasing colon weight and shortening colonic length.The mice from the damaged group appear splenomegaly due to inflammation and immune dysfunction.In the octacosanol group,the mental status of the mice was improved,a small amount or no blood in the stool was visible,and the DAI score was significantly reduced.Compared with the damage group,octacosanol significantly ameliorated of colonic ulcer,edema,bowel wall thickening,and increased intestinal weight and shorter length.Tissue sections were observed under a microscope.Compared with the normal group,the colon tissues of mice in the damage group showed significant acute inflammatory reaction symptoms such as mucosal erosion,ulcers,destroying of gland crypt structure,neutrophil infiltration and lost of goblet cells.In the octacosanol protective group mice,the above symptoms were obviously improved.Effects of octacosanol on the biochemical indexes of DSS-induced mice.Contents of myeloperoxidase(MPO),malondialdehyde(MDA)and nitric oxide(NO)in the colon tissues of the damaged group were 2.9,3.8 and 3.1 fold higher than that of the control group,respectively;which was significantly higher than that of the control group.Compared with the damaged group,the contents of MPO,MDA and NO in the colon tissues were decreased by 0.65,0.59 and 0.65 fold,respectively.The activity of MPO,and contents of MDA,NO in colonic tissues of mice treated with octacosanol were significantly decreased.Effects of octacosanol on expression of inflammatory factors in DSS-induced mice.The expressions of tumor necrosis factor-a(TNF-a),interleukin-1?(IL-1?),interleukin-6(IL-6)and inducible nitric oxide synthase(iNOS)were detected by real-time PCR and Western blot.Compared with the control group,the mRNA contents of four inflammatory factors in the colon tissue of the demaged group were increased by 7.51,6.16,12.95 and 16.54 fold,respectively.Compared with the demaged group,mRNA levels were reduced to 0.61,0.45,0.25 and 0.61 fold,respectively.The protein expression levels of TNF-?,IL-1?,IL-6 and iNOS in the colon tissues of the demaged group were 4.44,2.73,4.54 and 5.00 fold higher than that of the control group,respectively.While the four proteins in the octacosanol group were reduced to 0.75,0.63,0.78 and 0.80 fold,respectively.Octacosanol significantly down-regulated the expression of the above-mentioned inflammatory factor in mouse colon tissues.In this study,LPS(1.0 ?g/mL)was used to induce RAW264.7 cells produce inflammatory symptoms.Control,LPS + 10 ?g/mL,30 ?g/mL and 100 ?g/mL octacosanol group were set.The cells were observed under light microscope.The number of cells in each group was dense,the morphology was spindle-like and the growth in good condition,There was no significant difference in the cell status of each group.The effect of octacosanol on the activity of RAW264.7 cells was detected by MTS.There was no significant difference in the OD values between the control group and the octacosanol group(p>0.05).Compared with the control group,the other three groups had no significant difference(p>0.05).The results showed that 10-100 ?g/mL octacosanol solution had no effect on the growth and activity of RAW264.7 cells;it could be used in the follow-up study.Effects of octacosanol on expression of inflammatory factors in LPS-induced RAW264.7 cells.RT-PCR and Western blot was used to compare the expression of inflammatory genes in each group.Compared with the control group,the mRNA expression levels of TNF-a,IL-1?,IL-6 and iNOS in the LPS-induced group increased by 5.66,7.59,6.78 and 2.62 fold.Compared with the LPS group,10 ?g/mL,30?g/mL and 100 ?g/mL three groups of octacosanol-protected cells were significantly decreased in a dose-dependent manner.The protein expression of TNF-?,IL-1?,IL-6 and iNOS in the LPS-induced cells were 4.31,4.80,6.25 and 4.29 fold higher than those in the control group.Compared with LPS-induced cells,the expression levels of four proteins in the three groups of octacosanol protected group was significantly lower than that in LPS-induced group,and the difference was significant and dose-dependent.Octacosanol can significantly reduce the expression levels of inflammatory factors in LPS-induced RAW264.7 cells.Effects of octacosanol on the transcriptional activity of nuclear transcription factor-?B(NF-?B/p65)and activator protein-1(AP-1/c-Jun)in LPS-induced RAW264.7 cells.The transcriptional activity and its nuclear translocation were studied by using luciferase reporter gene and Western blot.The relative luciferase activity(RLU)of AP-1 and NF-?B in the LPS-induced group was significantly higher than that in the control group(3.41 and 2.49),while the RLU value in the octacosanol group was significantly decreased dependency.Compared with the control group,the expression levels of c-Jun and p65 protein in the cytoplasm of LPS-induced group increased by 2.17 and 1.56 fold,respectively.The expression of c-Jun and p65 protein in the cytoplasm was 0.53 and 0.41 folds.respectively.Compared with LPS-induced cells,the expression of c-Jun and p65 protein in the cells of octacosanol group was significantly decreased,and the expression of two proteins in the cytoplasm was significantly decreased in a dose-dependent manner,Octacosanol significantly inhibited the transcriptional activity and nuclear translocation of AP-1 and NF-?B in inflammatory cells.Phosphorylation of key target molecules in mitogen-activated protein kinase(MAPK)pathway in RAW264.7 cells by octacosanol.The phosphorylation of p38,c-Jun N-terminal kinase(JNK)and extracellular regulated protein kinases 1/2(ERK1/2)in LPS-induced cells was significantly higher than that in the control group(3.80,2.23 and 2.56,respectively).Compared with LPS cells,the phosphorylation of p38 and JNK in the cells of octacosanol group was significantly decreased in a dose-dependent manner.The expression of p-ERK1/2 in 100 ?g/mL octacosanol group was significantly decreased.These results suggested that LPS could promote the phosphorylation of p38,ERK1/2 and JNK target molecules in RAW264.7 cells and octacosanol could inhibit the level of phosphorylation in a dose-dependent manner.Effect of octacosanol and MAPK inhibitors on expressions of the TNF-?,IL-1?,p-p38,p-JNK,p-ERKl/2 protein and the activity of AP-1,NF-?B in RAW264.7 cells induced by LPS were evaluated by using western blotting and luciferase reporter assay.The results revealed the significantly decreased expression levels of TNF-?,IL-1?,p-p38,p-JNK protein in the MAPK inhibitors groups,and the AP-1,NF-?B transcription factor activity was also significantly reduced.The levels of inflammatory factors,MAPK phosphorylation and transcription factor activity were further reduced in the presence of octacosanol and MAPK inhibitors,which indicating that MAPK phosphorylation can increase the transcription factor activity and inflammatory factor expression.Octacosanol show the anti-inflammatory effects by inhibiting MAPK pathway.Comprehensive analysis,in the whole animal model,octacosanol could significantly improve the health status of DSS-induced colitis in mice,reduce the DAI score,reduce the damage of pathological tissue and decrease the activity of MPO and MDA and NO in colon.The expression levels of TNF-?,IL-1?,IL-6 and iNOS were down-regulated in colonic tissues.In the cellular model,octacosanol could down-regulate the expressions of TNF-?,IL-1?,IL-6,and iNOS genes,inhibit the nuclear translocation of p65 and c-Jun,attenuate NF-?B and AP-1 transcriptional activity,decrease the phosphorylation of p38,JNK and ERK1/2 in RAW264.7 cells induced by LPS.In addition,MAPK inhibitors significantly inhibited MAPK phosphorylation,inflammatory factor expression,and AP-1,NF-?B activity in the presence of octacosanol.It is suggested that the molecular mechanism of the anti-inflammatory mechanism of octacosanol is related to the inhibition of MAPK signaling pathway and the activation of NF-?B/AP-1,which results in the decreasing expression of inflammatory factors.