Mitigative Effect Of Chitosan Oligosaccharide On Acute And Chronic Stress Injury In Mice And Its Mechanism

The normal inflammatory response can protect tissues and cells from invading pathogens and foreign substances,whereas excessive inflammatory response may result in metabolic disorders and tissue injury.The inflammatory response is often accompanied by a large number of free radicals.Therefore,tissue and cell damage is closely related to inflammatory response and oxidative stress.In this study,the role and mechanism of chitosan oligosaccharide(COS)in alleviating acute hepatic and intestinal injury of mice were studied using lipopolysaccharide(LPS)to induce acute stress.Molecular mechanisms of COS in alleviating cellular inflammation and oxidative stress were studied using the mouse macrophage cells injury model constructed by LPS stimulation.The mitigative effect of chitosan oligosaccharide on high-fat diet(HFD)-induced obesity,adipose tissue abnormity,chronic hepatic injury and intestinal barrier dysfunction,and its mechanism were studied using HFD-fed mice.The main findings are as follows:Experiment 1.Mitigative effect of chitosan oligosaccharide on acute stress injury induced by lipopolysaccharide in mice and its mechanism(1)Optimization of chitosan oligosaccharides dose for alleviating lipopolysaccharide-induced acute stress injury in miceMice were received different doses of COS(0-800 mg/kg BW)by intragastric administration for 21 days,and then they were intraperitoneally injected with phosphate buffer saline(PBS,control group)or LPS.The results showed that 200-800 mg/kg B W COS reduced serum activities of AST,ALT,and LDH when compared with the LPS group.200mg/kg BW COS improved LPS-induced hepatic and renal edema,whereas 100 and 400 mg/kg BW COS only alleviated renal edema.Compared with the LPS group,intragastric administration of 400 mg/kg BW COS reduced MPO activities in the liver and spleen,whereas other doses of COS only reduced MPO activity in the spleen.200-800 mg/kg B W COS alleviated LPS-induced decrease of SOD activity and increase of MDA content in the liver.400 mg/kg BW COS has the best effect.The above results suggested that the optimal intragastric dose of COS for alleviating acute injury induced by LPS in mice is 400 mg/kg BW.(2)Mitigative effect of chitosan oligosaccharides on lipopolysaccharide-induced acute hepatic injury in miceForty-eight 5-week-old C57BL/6 male mice were divided into four groups:control,COS,LPS,and COS+LPS group.Mice were orally administered with either COS(400 mg/kg BW)or PBS for 30 days and then intraperitoneally injected with either 20 mg/kg BW LPS or PBS.The results showed that COS alleviated the increased serum levels of TNF-α,IL-1β and IL-6,and the increased activities of AST,ALT and LDH caused by LPS.COS also improved LPS-induced inflammatory cell infiltration and up-regulation of F4/80 protein levels in the liver.In addition,COS attenuated LPS-induced decrease of GSH-Px and SOD activities and increase of MDA and NO levels in the liver.These results suggested that COS could alleviate systemic inflammation as well as acute hepatic inflammation and oxidative stress injury caused by LPS.(3)Mitigative effect of chitosan oligosaccharides on lipopolysaccharide-induced acute intestinal injury in mice and its mechanismThe protective effect of COS on intestinal injury induced by LPS was studied using the aforementioned mouse model of acute injury.The results indicate that COS improved the mucosal morphology of jejunum and colon in LPS-challenged mice.COS reduced the LPS-induced down-regulation of jejunal tight junction protein and reduction of jejunal and colonic goblet cell numbers and mucin expressions.The mRNA expressions of anti-microbial peptides secreted by the jejunal and colonic cells were also up-regulated by COS.Additionally,COS decreased pro-inflammatory cytokines production and neutrophil recruitment in the jejunum and colon of LPS-treated mice.COS ameliorated jejunal and colonic oxidative stress through up-regulating the mRNA expressions of nuclear factor E2-related factor 2(Nrf2)and downstream antioxidant enzymes genes.Correlation analysis indicated that the beneficial effect of COS on intestinal barrier function was associated with its anti-inflammatory activities and antioxidant capacity.Experiment 2.Mitigative effect of chitosan oligosaccharide on Lipopolysaccharide-induced acute damage in mouse macrophage cells and its mechanism(1)Mitigative effect of chitosan oligosaccharide on acute stress damage induced by lipopolysaccharide in mouse macrophage cellsMouse macrophage cells(RA W264.7)were pretreated with different doses of COS(0-200 mg/kg B W)and then stimulated with LPS,the results showed that 50-200 μg/mL COS had no cytotoxicity to RAW264.7.50-200 μg/mL COS significantly alleviated LPS-induced up-regulation of TNF-α and IL-1β mRNA expressions,100 and 200 μg/mL COS significantly reduced IL-6 mRNA expression.50-200 μg/mL COS attenuated LPS-induced decrease of SOD activity and increase of MDA content,100 and 200 μg/mL COS significantly reduced NO level.The above results suggested that COS could alleviate LPS-induced inflammation and oxidative stress in RAW264.7 cells.(2)Mechanisms of chitosan oligosaccharides in reducing acute stress damage in mouse macrophage cellsRAW264.7 cells were divided into four groups:control,COS,LPS,and COS+LPS group.After pretreated with COS or PBS,LPS or PBS was added.The results showed that the mRNA expressions of NQO1,HO-1 and Gstal in the COS+LPS group were significantly higher than those in the LPS group.Moreover,the adenosine monophosphate-activated protein kinase(AMPK)phosphorylation levels in the COS group and COS+LPS group were significantly higher than those in the control group and LPS group.Inhibiting AMPK pathway with Dorsomorphin dihydrochloride(DD)eliminated the effect of COS on down-regulating the pro-inflammatory cytokine genes expressions and up-regulating the antioxidant enzyme genes expressions.The above results suggested that COS could alleviate the LPS-induced oxidative stress through up-regulating the expressions of antioxidant enzyme genes in RAW264.7 cells,and COS could attenuated the LPS-induced inflammation and oxidative stress by activating the AMPK signaling pathway,thereby reducing the acute injury induced by LPS in RAW264.7 cells.Experiment 3.Mitigative effect of chitosan oligosaccharide on high fat diet-induced chronic stress injury in mice and its mechanism.Forty 4-week-old C57BL/6 male mice were divided into four groups:normal chow diet(NCD)group,high-fat diet(HFD)group,HFD+low dosage of COS(HFDLC)group,and HFD+high dosage of COS(HFDHC)group.Mice were fed either a control diet or high-fat diet with or without COS(200 or 400 mg/kg BW)by oral gavage for 7 weeks.The results showed that intragastric administration of COS significantly inhibited the abnormal weight gain as well as increased serum lipid levels.COS reduced the fasting blood glucose increased by HFD and improved impaired glucose tolerance.COS attenuated adipocyte hypertrophy and inflammation caused by HFD.The hepatic lipid accumulation was significantly decreased by COS,which was attributed to decreased expressions of lipogenic genes and increased expressions of fatty β-oxidation related genes.In addition,pro-inflammatory cytokines,neutrophils infiltration and macrophage polarization were decreased by COS in the liver.Moreover,COS ameliorated hepatic oxidative stress through activating Nrf2 pathway and up-regulating gene expressions of antioxidant enzymes.Furthermore,COS administration up-regulated colonic tight junction protein expression,alleviated HFD-induced reduction of goblet cell number and down-regulation of mucin and antibacterial peptides expressions.These beneficial effects of COS on the liver and intestine were mediated by the activation of AMPK signaling pathway.In conclusion,COS alleviated acute hepatic inflammation and oxidative stress injury caused by LPS,and improved the intestinal barrier dysfunction induced by LPS in mice.COS inhibited LPS-induced inflammation and oxidative stress by activating AMPK signaling pathway in mouse macrophage cells,thereby reducing LPS-induced acute cell damage.COS attenuated obesity and adipose tissue abnormalities caused by HFD,and improved chronic hepatic and intestinal injury by activating the AMPK signaling pathway.