| Part ? Protective effects of tanshinone ?A sodium sulfonate on LPS-induced intestinal injury in miceObjective:To investigate the protective effects of tanshinone ?A sodium sulfonate on LPS-induced intestinal injury in mice.Methods:1.Six-week-old male C57BL/6J mice,were randomized into two groups:(1)LPS group,mice were challenged with LPS intraperitoneally 30 mg/kg(n=15);(2)TSS+LPS group,mice were pretreated with TSS(30 mg/kg),and 0.5h later received LPS 30 mg/kg intraperitoneally(n=15).The mortality of mice was observed every 12 hours.The survival of mice in 120h was statistically analyzed,and Kaplan-Meier survival curves were drawn.2.Six-week-old male C57BL/6J mice,were randomized into three groups:(1)Control group,mice received equal volume saline administration(n=30);(2)LPS group,mice were challenged with LPS 30 mg/kg intraperitoneally(n=30);(3)TSS+LPS group,mice were pretreated with TSS 30 mg/kg,and 0.5h later received LPS 30 mg/kg intraperitoneally(n=30).At 1,6,12 h after LPS injection,mice were sacrificed,and the jejunal and ileum was excised.The jejunal and ileum tissue injury was analyzed by HE staining.Results:Histological analysis showed the jejunum and ileum mucosa of the control group was intact and the villi were arranged neatly.While LPS induced severe edema and villus collapse,as well as infiltration of inflammatory cells into the jejunum and ileum mucosa at all observed time points.Length of jejunum and ileum villus was shortened at 1,6,12h after LPS stimulation compared with control group(P<0.05).In contrast,TSS treatment significantly reduced the edema and inflammatory cell infiltration of jejunum and ileum compared with the LPS group at all observed time points.TSS treatment at 1 h after LPS stimulation had no marked effect on the shortening of jejunum and ileum villus,however,from 6 h to 12 h,TSS treatment significantly inhibited the reduction of villus length compared with LPS group(P<0.05).The results showed that TSS could ameliorate LPS-induced intestinal pathological injury in mice.Pretreatment with TSS also increased the 48h survival rate by 40%and the 120h survival rate by 26.67%in LPS challenged mice(P<0.05).Conclusion:LPS can dramatically induce acute jejunal and ileum injury in mice,when compared with control mice.TSS treatment significantly reduced the villus edema and inflammatory cell infiltration and inhibited the reduction of villus length of jejunum and ileum compared with the LPS group.Meanwhile,TSS improved the survival rate of mice in LPS challenged mice(P<0.05).Part ? The influence of TSS on LPS induced inflammatory factors in intestinal injury in miceObjective:The present study is designed to explore the effect of TSS on LPS induced inflammatory factors in intestinal injury in mice.Methods:Six-week-old male C57BL/6J mice,were randomized into three groups:(1)Control group,mice received equal volume saline administration(n=30);(2)LPS group,mice were challenged with LPS 30 mg/kg intraperitoneally(n=30);(3)TSS+LPS group,mice were pretreated with TSS 30 mg/kg,and 0.5h later received LPS 30 mg/kg intraperitoneally(n=30).At 1,6,12 h after LPS injection,mice were sacrificed,and the jejunum was excised.Proinflammatory cytokine(TNF-?,IL-1?,and IL-6)in the jejunum tissue were examined by enzyme linked immunosorbent assay kit(ELISA)and Real-time PCR(RT-PCR).Anti-inflammatory factors(IL-10,TGF-P)in the jejunum tissue were examined by RT-PCR.Results:After LPS stimulation,both of mRNA and protein expressions of proinflammatory cytokine(TNF-?,IL-1 p,and IL-6)were markedly increased at all observed time points compared with control group(P<0.05).The mRNA expressions of inflammatory suppressing cytokine IL-10 and TGF-(3 were also promoted at 1,6,12 h after LPS injection(P<0.05).All the inflammatory cytokines were increased significantly at lh after LPS stimulation,and began to decline after LPS stimulation for 6 hours.TSS treatment significantly reduced the mRNA and protein expressions of TNF-?,IL-1?,and IL-6,and up-regulated the mRNA expressions of IL-10 and TGF-?,compared with LPS group(P<0.05).Conclusion:The protective effect of TSS on LPS-induced intestinal injury may be attributed to the inhibition of proinflammatory cytokine(TNF-?,IL-1?,and IL-6)and the up regulation of anti-inflammatory factors(IL-10 and TGF-?).Part III The effects of TSS on autophagy in LPS induced intestinal injury in miceObjective:To study the changes of autophagy in LPS-induced intestinal epithelial and the effects of TSS on autophagy in LPS induced intestinal injury in mice.Methods:Six-week-old male C57BL/6J mice,were randomized into three groups:(1)Control group,mice received equal volume saline administration(n=30);(2)LPS group,mice were challenged with LPS 30 mg/kg intraperitoneally(n=30);(3)TSS+LPS group,mice were pretreated with TSS 30 mg/kg,and 0.5h later received LPS 30 mg/kg intraperitoneally(n=30).At 1,6,12 h after LPS injection,mice were sacrificed,and the jejunum was excised.The expressions of autophagy markers(microtubule-associated light chain 3,LC-3 and Beclin-1)were detected by western blot and RT-PCR.The autophagosomes were also observed under electron microscopy,and the number of autophagosomes was also counted.Results:LPS stimulation decreased the jejunal epithelial autophagy levels as evidenced by reductions of both mRNA and protein expressions of Beclin-1 and LC-3,also the ratio of LC3-? to LC3-? compared with control group at 1,6,12 h(P<0.05).Pretreatment with TSS at early time point(lh)showed more reduction of autophagy levels,however,from 6 h to 12 h,TSS treatment significantly increased the mRNA and protein expressions of Beclin-1 and LC3,also the ratio of LC3-II to LC3-I,compared with LPS group(P<0.05).LPS group could not be observed any double or multiple membrane structures engulfed with cell organelle,which are key features of autophagy at all time points,however,TSS treatment group could find these structures at all time points.Conclusion:The protective effects of TSS on LPS-induced intestinal injury may be attributed to promotion of autophagy levels. |
PDF Full Text Request