| As a traditional Chinese medicine ingredient,?-mangostin has been proven to have anti-inflammatory,anti-bacterial and anti-oxidant effects.LPS,a major component of Gram-negative bacteria,can cause a variety of inflammations.In this paper,LPS was used to stimulate rat intestinal epithelial cells(IEC-6 cells)to construct an inflammatory model,and the anti-inflammatory effect of ?-mangostin and its potential molecular mechanism were discussed.Experiment one: The IEC-6 cell inflammatory model was established by using LPS as a stimulus source and cell viability was detected by CCK-8 method,cell morphology was observed by inverted microscope,and apoptosis was detected by flow cytometry.The results showed that the optimal concentration and time of LPS on IEC-6 cells was 10 ?g/mL for 24 h.The model conditions caused a decrease in cell viability,a change in cell morphology,and an increase in apoptotic rate(P < 0.05).Experiment two: Screening of the concentration of ?-mangostin by CCK-8 method and the morphological changes of the cells were observed under an inverted microscope,and the apoptosis was detected by flow cytometry.The results showed that the low,medium and high concentrations of ?-mangostin pretreated cells were 2.5,5,10 ?M,respectively.?-Mangostin can significantly reduce LPS-induced cell injury and apoptosis(P < 0.05).Experiment three: The secretion of PGE2,IL-6,TNF-? and IL-1? in the supernatant of the cells was detected by ELISA,and the secretion of NO was detected by Griess reagent.The mRNA levels of inflammatory factors iNOS,COX-2,IL-6,TNF-? and IL-1? were detected by qPCR.The expressions of TAK1,pTAK1,pIKK,I?B,pI?B,p65 and pp65 were detected by western blot.The results showed that ?-mangostin significantly inhibited the secretion of NO,PGE2,IL-6,TNF-? and IL-1? induced by LPS(P < 0.05).At the same time,the expression of iNOS,COX-2,IL-6,TNF-? and IL-1? mRNA induced by LPS was also inhibited(P < 0.05).?-Mangostin attenuates the decrease in I?B protein expression induced by LPS and inhibits the expression of pTAK1,pIKK,pI?B and pp65 proteins induced by LPS(P < 0.05).Immunofluorescence results showed that ?-mangostin could significantly inhibit p65 entry into the nucleus caused by LPS.The results indicate that ?-mangostin can attenuate LPS-induced inflammation by inhibiting the TAK1/NF-?B signaling pathway.Experiment four: Whole genome sequencing was performed using the Illumina hiseq sequencing platform and the results were verified by qPCR.A total of 37199 probes profiled were detected,928 genes were significantly up-regulated after LPS induction,and 1068 genes were significantly down-regulated(log2 fold change > 1 or log2 fold change <-1,P < 0.05).After pretreatment with ?-mangostin,151 genes were significantly down-regulated,and 324 genes were significantly up-regulated(log2 fold change > 1 or log2 fold change <-1,P < 0.05).We found that GO,which is significantly enriched by differential expressed genes,is mainly associated with inflammation and oxidative stress,and is also associated with apoptosis.From pathway analysis we found that differentially expressed gene enrichment is closely related to NF-?B signaling pathway and inflammatory bowel disease(IBD).Experiment five: LPS was used to construct a rat enteritis model to investigate the protective effect of ?-mangostin on the intestinal tract of rats.The results showed that ?-mangostin could effectively improve the intestinal damage caused by LPS,especially for intestinal villi.The above results show that ?-mangostin has a good anti-inflammatory effect and can be used as an option for the treatment of gastrointestinal inflammation. |
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