| Purpose:To explore the mechanism of Yangwei Jiedu Mixture(YJM)on intestinal mucosa against sepsis by observing the protective effects of YJM combined with TIENAM to intestinal mucosa in rats,by focusing on such indicators as inflammation,apoptosis and intestinal mucosal barrier.Material and method:140 rats were randomly divided into seven groups,control group,model group,regular dose group,high dose group,TIENAM group,TIENAM plus regular dose group and TIENAM plus high dose group.(20 rats each group)The end of cecum were ligated and punched to set up septic rat model,(6.3 times of dosages of human use body weights),all the rats were treated with different factors intervention respectively as following,NS(1ml/100 g,po,bid)for Control group and model group,Yangwei Jiedu(1.4g/100 g,po,bid)for regular dose group,Yangwei Jiedu(2.8g/100 g,po,bid)for high dose group,Yangwei Jiedu(0.7g/100 g,po,bid)for low dose group,Tienam(100mg/kg,ip,bid)for TIENAM group,Tienam(100mg/kg,ip,bid)and Yangwei Jiedu(1.4g/100 g,po,bid)for TIENAM plus regular dose group,Tienam(100mg/kg,ip,bid)and Yangwei Jiedu(2.8g/100 g,po,bid)for TIENAM plus high dose group,Tienam(100mg/kg,ip,bid)and Yangwei Jiedu(0.7g/100 g,po,bid)for TIENAM plus low dose group.After 2 days administration,death rate was calculated and serum and ileum were harvested.Experiment 1: The content of TNF-? and IL-1? in the serum of each group were detected by enzyme-linked immunosorbent assay(ELISA).The HE staining of intestinal tissue of rats was used to observe the morphology of ileum specimens.Experiment 2: ELISA was used to obseved the content of TNF-? and IL-1? protein in intestinal tissue of rats.Real-time quantitative PCR(Real-time PCR)was used to examine the m RNA expressions of TNF-??IL-1? in intestinal tissue of rats in each group.Western blotting was used to compare the expressions of TLR2/4,NF-?B,Bcl-2,Bax and caspase-3 protein in intestinal tissue.Experiment 3: Immunohistochemical was used to detect the expression and distribution of Occludin,Claudin-1 and ZO-1 protein in intestinal tissue of rats.Western blotting was used to compare the protein expressions of Occludin,Claudin-1 and ZO-1 in intestinal tissue.Real-time quantitative PCR was used to examine and compare the m RNA expressions of occludin,Claudin-1 and ZO-1 between proups.Results: 1.The general status of rats,survival rate and ileal tissue morphological changes.1.1 General status The control group: The fur,mental state,reaction sensitivity,activity status,behavior,food intake,water intake and stool excretion of control group rats were all normal and body weight increased rapidly.The Model group: The fur condition was dry and hairless,apathetic,poor in stress,decreased in activity,curled up in the back of the arch,like curled up together,decreased food intake,poor wound healing and red eyes;The regular dose group and the high dose group: A slight dry fur color and slight mild malaise.The reaction and activity were still curled up.The food intake and water intake were both increased compared with the model group.The degree of wound healing still,loose stools and red eyes;TIENAM group and TIENAM plus regular dose group: Fur condition slight dry coat color,mild malaise,moderate reaction and activity,curled up sometimes,and their food intake and water intake increased compared with model group.Surgical wounds Basic healing,stool slightly thin,red eyes;TIENAM plus high dose group: Slightly dry fur,no apathetic,the reaction and activity were basically normal,occasional crouching,eating and drinking water were increased than the model group,surgical wound healing,thin stools slightly and normal eye condition.1.2 Death rate Zero deaths in control group,12 deaths in Model group(60%),11 deaths in regular dose group(55%),11 deaths in high dose group died(55%),10 deaths in TIENAM group(50%),11 deaths in TIENAM plus regular dose group(55%)and 8 deaths in TIENAM plus high dose group(40%).1.3 Histomorphology of ileum Control group: normal intestinal cell morphology.Model group: there were severe edema of intestinal cells,a lot of inflammatory cell infiltration,and necrotic epithelial cells.Regular dose group,high dose group,TIENAM group,TIENAM plus regular dose group and TIENAM plus high dose group inflammatory cells decreased,edema reduced,no necrotic epithelial cells,in which high dose group and TIENAM plus high dose group are particularly evidenced.2.Factors involved in TLR2/4-NF-?B signaling pathways in intestinal tissue 2.1 The changes of intestinal tissue TNF-? and IL-1? 2.1.1 TNF-? and IL-1? content in intestinal tissue Compared with the control group,the levels of TNF-? and IL-1? in the model group were significantly increased(P <0.01).Compared with the model group,the levels of TNF-? in TIENAM group,TIENAM plus high dose group were significant decline(P<0.01),the levels of IL-1? in each treatment group were significant decline(P <0.01).Compared with the TIENAM group,the levels of TNF-? in TIENAM plus high dose group were significantly increased(P <0.01),the levels of IL-1? in the TIENAM plus high dose group and TIENAM plus regular dose group were significantly decreased(P<0.01),TIENAM plus high dose group were more significantly lower.2.1.2 TNF-?,IL-1?m RNA expression in rat intestinal tissue Compared with the control group,the expression of TNF-? and IL-1? m RNA in the model group was significantly increased(P <0.01).Compared with the model group,the expression of TNF-? m RNA in each treatment group was significantly increased(P<0.01).The expression levels of IL-1? m RNA in TIENAM group and TIENAM plus high dose group were significantly decreased(P <0.01).Compared with TIENAM group,the levels of TNF-? and IL-1? m RNA in TIENAM plus high dose group were all decreased,but there was no statistical significance(P >0.05).2.2 TLR2,TLR4 and NF-?B protein expression intestinal tissue Compared with the control group,the level of TLR2 protein in the model group was significantly increased(P<0.01),the levels of TLR4 and NF-ebb protein were increased(P <0.05),which were statistically significant.Compared with the model group,The expression of TLR2,TLR4 and NF-?B protein in intestinal mucosa of rats in each treatment group were significantly decreased(P<0.05);Compared with TIENAM group,the lever of TLR2 in regular dose group?TIENAM plus regular dose group and TIENAM plus high dose group were decreased(P<0.05).The expression of NF-?B protein in TIENAM plus high dose group was decreased(P<0.05).3.Protein expression of apoptosis-related factors Compared with the control group,the expression of Bax and Capase3 protein in model group was significantly increased(P<0.01),while the expression of Bcl-2 was significantly decreased(P<0.01),which were statistically significant.Compared with the model group,the expression of Bax and Caspase3 protein in the intestinal tissue of rats in the TIENAM plus high dose group were significantly decreased,while the levels of Bcl-2 protein were significantly decreased(P<0.01);Compared with the TIENAM group,the expression of Bax and Capase3 protein in TIENAM plus high dose group significantly decreased(P<0.01),while the expression of Bcl-2 was significantly increased(P <0.01).4.The changes of intestinal mucosal barrier related proteins 4.1 IHC data of the expression and distribution of rat intestinal mucosal barrier related protein 4.1.1The expression and distribution of Occludin,Claudin-1 and ZO-1 Light microscope showed that a large number of positive brown granules were observed around the intestinal cell membrane of Occludin,Claudin-1 and ZO-1 proteins in control group,which were ordered in an orderly manner.Compared with the control group,Occludin expression were decreased,the order of arrangement was a little confused.Compared with the model group,Occludin expression was significantly increased and arranged in an orderly manner.TIENAM plus high dose group was more obvious.4.1.2 The expression of gray value calculation The integral optical density of Occludin: Compared with the control group,the integral optical density of the model group was significantly decreased(P <0.01).Compared with the model group,the integral optical density of TIENAM group,TIENAM plus regular dose group and TIENAM plus high dose group was significantly increased(P <0.01).Compared with the TIENAM group,the integral optical density of TIENAM plus regular dose group and TIENAM plus high dose group was significantly increased(P<0.01).The integral optical density of Claudin-1: Compared with the control group,the model group was significantly decreased(P<0.01).Compared with the model group,except for the regular dose group group,the other treatment groups were significant increased(P<0.01).Compared with the TIENAM group,TIENAM plus regular dose group and TIENAM plus high dose group was significantly increased(P<0.01).The integral optical density of ZO-1: Compared with the control group,the integral optical density of the model group was significantly decreased(P<0.01).Compared with the model group,the integral optical density of each treatment group was significantly increased(P<0.01).4.2 m RNA expression of intestinal barrier function-related gene Compared with the control group,the m RNA expressions of Occludin,Claudin-1 and ZO-1 in model group were significantly decreased(P<0.01).Compared with model group,the m RNA expressions of Occludin,Claudin-1 and ZO-1 in high dose group,TIENAM group and TIENAM plus high dose group were significantly increased(P<0.01),of which TIENAM plus high dose group had the most significant changes,while the normal dose group did not change significantly.Compared with the TIENAM group,the m RNA expression of Occludin in TIENAM plus high dose grou had an increasing trend,but the difference was not significant,m RNA expression levels of Claudin-1 and ZO-1 were significantly increased(P<0.01),Occludin m RNA expression was significantly decreased in normal dose group(P<0.05).4.3 Protein expression of barrier function-related factors Compared with the control group,the expressions of Occludin,Claudin-1 and ZO-1 in model group were significantly decreased(P<0.01).Compared with the model group,the expression of Occludin,Claudin-1 and ZO-1 in each treatment group was significantly increased(P<0.01),of which TIENAM plus high dose group was the most obvious,and high dose group was significantly more than regular dose group.Compared with the TIENAM group,the Claudin-1 protein expression of high dose group and regular dose group were significantly lower(P<0.01),the expression of Occludin,Claudin-1 and ZO-1 protein in TIENAM plus high dose group was significantly increased(P<0.01),the expression of Occludin and ZO-1 protein in TIENAM plus regular dose group was significantly increased (P<0.01).Conclusion: 1.Yangwei Jiedu mixture combined with TIENAM can obviously protect intestinal mucosa of sepsis model rats,of which high dose plus TIENAM group is excellent,indicating that high-dose Yangwei Jiedu mixture combined with TIENAM can play synergistic effect.2.Yangwei Jiedu mixture can reduce the inflammatory response,by regulating inflammatory factors.3.Yangwei Jiedu mixture can reduce the apoptosis of intestinal epithelial cells by regulating the related apoptosis factors.4.Yangwei Jiedu mixture can protect the intestinal mucosa by up-regulating the expression of intestinal mucosal barrier proteins. |
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