The Protective Effect And Mechanism Of Tong-fu-li-fei Decoction On Intestinal Function Injury In Sepsis

ObjectiveThe clinical and basic research were carried out to explore the protective effect and mechanism of Tong-fu-li-fei Decoction(TFL)on intestinal function injury in sepsis.Methods1.Clinical ResearchThis study retrospectively collected the clinical data of patients with sepsis intestinal function injury who were hospitalized in the Department of critical care medicine,the First Affiliated Hospital of Guizhou University of traditional Chinese medicine from March 2016 to September 2019.According to the application of Tong-fu-li-fei decoction,the patients were divided into TFL group and NTFL group.The baseline data of patients and the data of 7 days after admission to ICU were recorded,including the information of gender,the basic vital signs,the mechanical ventilation was used or not,the infection index,the organ dysfunction index,the intestinal function injury index,the duration of mechanical ventilation,the severity and prognosis and others.spss 20.0 statistical software was used for the statistical analysis,and ordered multi classification logistic regression model was used to detect independent risk factors of intestinal function injury,all analysis use bilateral test,P < 0.05 was statistically significant.2.Basical ResearchThe Sprague-Dawley(SD)rats were divided into the sham group,model group,the 3.6 g/kg TFL-treated group(3.6g/kg TFL)and 7.2 g/kg TFL-treated group(7.2g/kg TFL).The sepsis model was induced by cecal ligation and puncture(CLP).Immediately following the surgery,TFL-treated group were received with TFL decoction by gavage at a dose of 3.6 or 7.2 g/kg body weight per day for7 days.Sham group and CLP(model)group were given distilled water(10 ml/kg)once a day by gavage.The blood and ileum were taken for detection.The levels of TNF-?,IL-1?,IL-6 and IL-10 cytokines were detected by enzyme-linked immunosorbent assay(ELISA),the hematoxylin and eosin(HE)staining was used to observe the pathological changes of intestine,Real-time quantitative PCR was used to detect the expression of zonula occludens proteins-1(ZO-1),claudin-1 and occludin m RNA in ileum tissue,the protein levels of ZO-1,occludin and claudin-1 were detected by immunohistochemistry and Western blotting analysis,spss20.0 was used to do statistical analysis,Prism 8.0(graphpad software)is used for statistical mapping.all analysis use bilateral test,P < 0.05 was statistically significant.Results??Clinical Research1.The comparison of baseline data between the two groups showed that the respiratory rate of TFL group was significantly higher than that of NTFL group,and the pulse oxygen saturation was significantly lower than that of NTFL group(P < 0.05);there was no significant difference in Acute Physiology and Chronic Health Evaluation(APACHE II)score,Sequential Organ Failure Assessment(SOFA)score and Intra-abdominal Pressure(IAP)between the two groups(P >0.05).7 days after treatment,the data showed that the pulse oxygen saturation in TFL group was significantly higher than that in NTFL group,while APACHE II score and IAP were significantly lower than that in NTFL group(P < 0.05).There was no significant difference in respiratory rate and SOFA score between the two groups(P > 0.05).2.There was no significant difference in the baseline grade of the acute gastrointestinal injury between the two groups(P > 0.05);there was significant difference in the baseline grade of the acute gastrointestinal injury between the two groups on the 7th day after treatment,and the proportion of grade I and grade II in TFL group was higher(P < 0.05).3.The ordinal logistic regression analysis model was established with the grade of acute gastrointestinal injury in 7 days after treatment as the dependent variable.After correction of mean arterial pressure,body temperature,white blood cell,APACHE II score,SOFA score and PCT,the results showed that the probability of increase of grade of acute gastrointestinal injury in patients with rectal drip of Tong-fu-li-fei decoction was 0.30 times higher than that of patients without rectal drip of Tong-fu-li-fei Decoction(OR = 0.30,P = 0.015).4.Compared with NTFL group,the duration of mechanical ventilation and the length of stay in ICU in TFL group were significantly higher than those in NFTL group(P < 0.05).There was no significant difference in 28-day mortality between the two groups(P > 0.05).??Basical Research1.The survival rate of CLP induced sepsis rats in TFL group was significantly higher than that in model group(P < 0.05).2.Compared to the level of plasma TNF-a and IL-1? of the sham group,the level of TNF-a and IL-1? was significantly higher in the model group(P <0.05)and reduced to a comparable level in the 3.6 g/kg and 7.2 g/kg TFL group(P < 0.05).The levels of TNF-a,IL-1? and IL-6 of ileum tissue in the sham group were significantly lower than that in the model group(P < 0.05),3.6 g/kg and 7.2 g/kg TFL group(P < 0.05).The levels of TNF-a,IL-1? and IL-6 of ileum tissue in the 3.6 g/kg and 7.2 g/kg TFL group were significantly lower than that in the model group(P < 0.05,P < 0.01).For the level of IL-10 of ileum tissue,there was a significantly higher level in the model group and in the 3.6 g/kg and 7.2 g/kg TFL group compared to the level of IL-10 in the sham group(P < 0.05).Unlike other factors,the model group had statistically significantly lower IL-10 level than that in the 3.6 g/kg and 7.2 g/kg TFL group(P < 0.05,P < 0.01).3.Histopathological results showed that the ileum tissue structure was intact and the intestinal villi were arranged neatly in the sham operation group under light microscope,the surrounding vascular structure was normal,there was no obvious bleeding,the muscle fibers arranged in order,the serous membrane was normal.In the model group,the intestinal mucosa was destroyed,edema and degeneration of intestinal mucosal epithelial tissues were observed,intestinal villi were seriously damaged and disordered,mucosal swelling and bleeding were observed around the blood vessels,epithelial cells were separated from the lamina propria,and hemorrhage,edema and necrosis were observed in the lamina propria,there were a large number of proliferation of lymphocytes,neutrophils were observed.After treatment,moderate mucosal swelling and bleeding were observed around the blood vessels in the 3.6 g /kg TFL treatment group and 7.2 g / kg TFL treatment group,The mucosal tissue edema,hemorrhage and necrosis of the small intestine of the rats in the administration group were significantly alleviated,the epithelial mucosa was slightly edema,the villi of intestinal mucosa were moderately damaged,the top part of villi was destroyed,the basic villi structure remained intact,the lamina propria was slightly edema,hemorrhage and necrosis were not obvious,and the results were basically intact,and a small number of proliferative lymphocytes and Neutrophils were observed.4.The results showed that relative expression levels of ZO-1?Claudin-1and occludin genes in the ileum tissues of the model group were significantly lower than those of the sham group(P < 0.01).Compared with the model group,the relative expression levels of ZO-1?Claudin-1 and occludin genes in the TFL treatment group were significantly increased(P < 0.01).5.As shown by immunohistochemistry,the ZO-1 protein expression in model group(IOD: 2549 ± 786)was lower than that of sham group(IOD: 15,809 ± 1566);the Occludin protein expression in model group(IOD: IOD: 1951±845)was lower than that of sham group(IOD: IOD:14088 ± 1808);the Claudin-1 protein expression in model group(IOD: 616.3±161.8)was lower than that of sham group(IOD: 6572±704).TFL-treated rats(IOD: 3.6 g/kg TFL,5089 ± 846;7.2 g/kg TFL,7094 ± 1465)exhibited increased ZO-1 in ileal tissue compared with the rats in model group.7.2 g/kg TFL treatment upregulated the expression of occludin(IOD: from 1951 ± 845 to 6632 ± 704)and claudin-1(IOD: from 616± 162 to 4385 ± 671)protein compare with the model group.6.Western blot was used to detect the protein levels of ZO-1,occludin and Claudin-1 in intestinal tissue of rats.The results showed that compared with the sham operation group,the expression of ZO-1,occludin and Claudin-1in intestinal tissue of model group was significantly decreased(P < 0.05,P < 0.01).Compared with the model group,the expression levels of ZO-1,occludin and Claudin-1 in colorectal tissue of 3.6 g / kg TFL treatment group and 7.2 g / kg TFL treatment group were significantly increased(P < 0.05,P < 0.01).ConclusionTong-fu-li-fei decoction can reduce the intra-abdominal pressure and intestinal function injury grade,reduce the APACHE II score and improve the prognosis of patients with sepsis.Its rectal drip is an independent protective factor of intestinal function injury,and has a certain protective effect on intestinal function injury in sepsis.Tong-fu-li-fei decoction can improve the survival rate of rats with sepsis induced by CLP,reduce the intestinal mucosal damage caused by sepsis,reduce the expression of TNF-?,IL-1? and IL-6 proinflammatory cytokines,increase the expression of IL-10 anti-inflammatory cytokine,reduce the inflammatory reaction,and restore the expression of tight junction proteins and m RNA of occludin?Claudin-1 and ZO-1 in the intestinal tissue of CLP-induced sepsis rats.In addition,it can improve the intestinal barrier function by reducing the inflammatory reaction and upregulating the expression of occludin,claudin-1 and ZO-1,which may become a new method for the treatment of intestinal dysfunction in sepsis.