| Objective:5-Fluorouracil(5-FU)is a commonly used antitumor drug.Its main side effect is to induce intestinal mucositis and lead to the secretion of a large number of proinflammatory cytokines.Almost all patients treated with 5-FU will develop intestinal mucositis,which is an important factor causing the interruption of tumor treatment and directly leading to the failure of tumor treatment.At present,there are still no effective prevention and treatment methods.Short chain fatty acids(SCFAs)are important anti-inflammatory metabolites of intestinal flora and the main regulatory factor of "intestinal-immune axis".They have the functions of inhibiting immune cell activation,promoting intestinal epithelial cell proliferation and maintaining the integrity of intestinal mucosal barrier.In this study,prebiotic inulin was used to regulate intestinal flora and increase SCFAs production in mice.Based on metabolomics technology,the mechanism of inulin alleviating intestinal mucositis induced by 5-FU in mice through "intestinal-immune axis" pathway was analyzed;Further study the mechanism of three major SCFAs,namely NaAc,NaPc and NaB,inhibiting the inflammatory response of THP-1 cells and Caco-2 cells induced by5-FU and maintaining the integrity of Caco-2 cell tight junction in vitro.Methods:1.The intestinal mucositis model of ICR mice was established,and the normal control group,5-FU group and inulin group were set up.1%(m/v)inulin solution instead of drinking water(pretreatment for 30 days),intraperitoneal injection of 5-FU(30 mg/kg)for5 consecutive days induced intestinal mucositis.After the last administration for 24 h,the serum,spleen tissue and stomach to cecum tissue of mice were taken.The intestinal length of mice was measured to evaluate the intestinal injury;the expressions of NLRP3,IL-10,IL-1β and s Ig A were determined by q RT-PCR and ELISA;the pathological changes of intestinal mucosa were observed by HE staining;the expressions of tight junction protein ZO-1 and occludin in mouse small intestinal mucosa were detected by IHC.The changes of metabolites in serum were detected by UPLC-QTof-MS/MS.The concentrations of three major SCFAs in serum were determined by LC-MS/MS.2.THP-1 cells were pre-treated with 100 μmol/L of NaAc,NaPc and NaB for 24 h,then 2.5 mmol/L 5-FU for 24 h.The m RNA expressions of NLRP3,Caspase-1,IL-1β,IL-6,and IL-10 were determined by q RT-PCR.The ROS in THP-1 cells was determined by DCFH-DA fluorescent probe.The expressions of LC3-Ⅱ,Beclin-1 and NF-κB p65 pathway activation were determined by Western blotting.Apoptosis rate was determined by Annexin V-FITC/PI.Cell metabolomics were determined by UPLC-QTof-MS/MS.3.Caco-2 cells were pre-treated with 100 μmol/L of NaAc,NaPc,and NaB for 24 h,then 5 mmol/L 5-FU for 24 h.The m RNA expressions of NLRP3,Caspase-1,IL-1β,and IL-18 were determined by q RT-PCR;the expressions of NLRP3,Caspase-1,GSDMD and NF-κB p65 pathway activation were determined by Western blotting;The ROS was determined by DCFH-DA fluorescent probe;the m RNA expressions of tight junction proteins ZO-1,Occludin and MUC2 were determined by q RT-PCR.Results:1.After intraperitoneal injection of 5-FU,the concentrations of three main SCFAs in mouse serum decreased significantly(P < 0.05).Oral inulin could significantly increase the concentrations of the three main SCFAs in mouse serum and improve the disorder of serum metabolomics(P < 0.05).The expressions of NLRP3 in spleen and IL-1β in serum of 5-FU group were significantly higher than those in the normal control group,and the expressions of IL-10 and s Ig A were significantly lower(P < 0.05).Oral inulin can decrease the expressions of NLRP3 and IL-1β,and increase the expressions of IL-10 and s Ig A(P <0.05).HE staining showed that the injury of intestinal mucosa in the inulin group was significantly improved than that in the 5-FU group.IHC results showed that the expressions of tight junction protein ZO-1 and Occludin in the inulin group were significantly higher than those in the 5-FU group(P < 0.05).2.When treated with 5-FU,vompared with the normal control group,the m RNA expressions of NLRP3,caspase-1,IL-1β and IL-6 in THP-1 cells were significantly increased(P < 0.05);the expressions of autophagy related proteins Beclin-1 and LC3-Ⅱwere also significantly increased(P < 0.05);the NF-κB p65 expression were significantly increased in the nucleus(P<0.05);the ROS expression and cell apoptosis rate increased significantly(P < 0.05).Compared with the 5-FU group,the three main SCFAs can inhibit the m RNA expressions of NLRP3,Caspase-1 and IL-6 in THP-1 cells(P < 0.05),and increase the m RNA expression of IL-10(P<0.05),and inhibit the expressions of autophagy related proteins Beclin-1 and LC3-Ⅱ(P < 0.05);the expression of NF-κB p65 in the nucleus decreased significantly in the NaAc and NaB groups(P < 0.05);the expressions of ROS and IL-1β were significantly decreased in the NaPc and NaB groups(P < 0.05),and cell apoptosis rate were significantly decreased in the NaAc group(P < 0.05).The results of non-targeted metabolomics showed that 5-FU could induce the metabolic disorder of THP-1 cells,and the intervention of three main SCFAs can significantly improve the cellular amino acid metabolism,glycerol phospholipid metabolism and sphingolipid metabolism.3.When treated with 5-FU,compared with the normal control group,the expressions of NLRP3,Caspase-1,IL-1β,IL-18,GSDMD,ROS and nucleus NF-κB p65 in Caco-2cells were significantly increased(P < 0.05).Compared with 5-FU group,the three main SCFAs can inhibit the m RNA expressions of NLRP3,Caspase-1,IL-1β and IL-18 in Caco-2 cells(P < 0.05),and decrease the production of ROS and the expression of NF-κB p65 in the nucleus(P < 0.05),and inhibit the expression of GSDMD protein(P <0.05),and significantly up-regulated the m RNA expressions of Occludin and MUC2 in Caco-2 cells(P < 0.05).Conclusion:Oral inulin can significantly increase the products of three main SCFAs(NaAc,NaPc and NaB)in serum,improve the disorder of serum metabolomics,protect the integrity of intestinal mucosal barrier via inhibiting the activation of NLRP3 inflammasome. |
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