| Purpose:Evaluate the efficacy and safety of Fufang Zhixue Powder in preventing ICU stress ulcer bleeding through clinical trials.Take stress ulcer model rats as the research object,and preliminarily clarify the mechanism of traditional Chinese medicine in the"intestinal microbiota-metabolite-host"interaction through experimental research.Methods:1.By conducting a prospective,multicenter,randomized controlled trial（RCT）,a total of 270 patients with stress ulcer risk factors and stress sources were recruited from the intensive care unit（ICU）of 9 hospitals,including Hospital of Chengdu University of TCM,Sichuan Second TCM Hospital,Chengdu Shuangliu Hospital of TCM,Chengdu Longquanyi Hospital of TCM,Chengdu Xindu Hospital of TCM,Chengdu Xinjin Hospital of TCM,Chengdu Jintang Hospital of TCM,Leshan Hospital of TCM,Shehong Hospital of TCM.Patients were randomly and equally assigned to the experimental group and the control group.Both groups received routine Western medicine treatment.Then,the experimental group was treated with Fufang Zhixue Powder,while the control group was treated with a placebo.Both groups were treated for 7 days.Main measures were bleeding incidence and 28-day all-cause mortality,while secondary measures were APACHE II score,SOFA score,gastrointestinal function classification score,abdominal pressure,and ICU stay length.Safety indicators,such as liver and kidney function,were also evaluated to assess the efficacy and safety of Fufang Zhixue Powder in preventing stress ulcers bleeding.2.SD rats were adapted to a new diet for one week and divided into blank control group（C）,model control group（M）,positive control group（P）,and Chinese medicine prevention group（Z）.All rats were given the corresponding drugs or physiological saline by continuous gavage for 7 days.After the 7th gavage,rats were fasted for 24hours and then placed head-up in an electric constant temperature water bath（19±1）℃for 30 minutes to prepare a water immersion-restraint stress ulcer animal model.24hours later,the rats were sacrificed,and the abdominal aorta blood,the gastric tissue and ileum contents of the rats were collected for measurement.The gastric ulcer index of gastric tissue was evaluated according to the Guth standard;HE staining was used to observe the pathological changes of gastric tissue;QPCR technology was used to detect the mRNA expression levels of serum inflammatory markers（TLR4,MyD88,NF-κB p65）;and 16S rDNA was used to sequence the intestinal contents of each group of rats.The richness and diversity of microbial communities in each group of rats were analyzed,and the relative abundance of bacterial populations was analyzed at the phylum and genus levels.At the same time,non-targeted metabolomics methods were used to analyze the serum metabolites of each group of rats from the perspective of host metabolism.KEGG enrichment analysis was performed.Furthermore,Pearson correlation analysis was applied to analyze the bacterial classification group and the differential serum metabolites to preliminarily clarify the mechanism of Chinese medicine in the"intestinal microbiota-metabolites-host"interaction.Results:1.Clinical trial results（1）A total of 270 patients were included in the clinical trial research,30 dropped out,and finally 240 patients were included in the statistical analysis.Stressors were:cardiovascular and cerebrovascular accidents,traumatic brain injury/trauma/sepsis,MODS,shock,and post-CPR.（2）Main efficacy indicators:There was no statistically significant difference in the28-day all-cause mortality rate between the experimental and control groups（16.00%vs 15.65%,P>0.05）;the bleeding rate in the experimental group was lower than that in the control group（20.00%vs 33.91%,P<0.05）,and the difference was statistically significant.（3）Secondary efficacy indicators:ICU hospitalization days in the experimental group were lower than those in the control group[（13.53±5.02）d vs（15.40±6.20）d,P<0.05];APACHE II score in the experimental group was lower than that in the control group[（12.39±5.35）vs（15.10±5.86）,P<0.05];SOFA score in the experimental group was lower than that in the control group[（3.31±3.02）vs（5.28±4.55）,P<0.05].However,there was no statistically significant difference in the intra-abdominal pressure[（4.67±3.65）cm H2O vs（5.10±3.82）cm H2O,P>0.05]and the gastrointestinal function classification score（1.18±1.18 vs 1.34±1.29,P>0.05）between the two groups.（4）There was no significant difference in safety indicators between the two groups of patients.Animal experiments results:（1）Gastric ulcer index:The gastric ulcer index of rats in C group was lower than that of M group（17.17±3.17 vs.25.00±2.10,P<0.05）.（2）Gastric tissue pathological damage:The mucosal layer of rats showed multifocal necrosis,with a small amount of yellow substance deposition within the necrotic area,and with dilation of capillaries and infiltration of inflammatory cells around the necrotic area in Z group.Pathological damage was less than M group.（3）Serum inflammatory indicators:Compared with M group,C group showed a decrease in the expression of serum NF-κB p65 mRNA（0.836±0.245 vs.2.650±1.070,P<0.05）,but no significant difference was observed in the expression of serum TLR4mRNA（0.920±0.056 vs.1.033±0.221,P>0.05）or MyD88 mRNA（1.027±0.070 vs.1.373±0.505,P>0.05）.（4）High-throughput sequencing analysis of 16S rDNA of intestinal microbiota:（1）At the phylum level,compared to M group,the Chinese herbal medicine reduced the ratio of Firmicutes to Bacteroidetes（F/B）;（2）At the genus level,the Chinese medicine treatment reduced the relative abundance of Cetobacterium,while significantly enriching the Lactobacillus;（3）LEfSe results showed that the probiotics such as Lactobacillus、Psychrobacter、Aerococcus、Allobaculum、Faecalitalea were significantly enriched in Z group.（5）Non-targeted plasma metabolomics results:（1）There was a significant difference between C and M groups,indicating significant changes in rat plasma metabolites and physiological metabolism disorders.（2）Compared with M group,180metabolites were considered potential biomarkers in Z group,among which the concentrations of glycyrrhetinic acid,pyrophosphate,fusidic acid,and 10-nitro-oleic acid were significantly increased（P<0.05 and FDR<0.05）.（3）KEGG enrichment results of differential metabolites showed that Fufang Zhixue Powder may be related to lysine degradation,phenylalanine metabolism,acyl-biosynthesis,arachidonic acid metabolism,steroid hormone biosynthesis,vitamin B6 metabolism,digestion and absorption of vitamins,steroid biosynthesis,and arginine and proline metabolism.（6）Correlation analysis between gut microbiota and metabolomics:With the intervention of Fufang Zhixue Powder,there was a strong correlation between some gut microbiota and differential metabolites.The core intestinal microbiota includes Allobaculum and Faecalitalea,and is positively correlated with the production of short-chain fatty acids,which can inhibit oxidative stress and inflammatory reactions.Conclusion:1.Fufang Zhixue Powder,when applied together with Western medicine routine prevention methods,can reduce the incidence of bleeding for patients with stress ulcers in ICU,shorten ICU stay length,reduce APACHE II score and SOFA score,but there is no significant benefit in improving the 28-day mortality rate,intra-abdominal pressure,and gastrointestinal function grading score of patients.2.Fufang Zhixue Powder can reduce the ulcer index of gastric tissue in water immersion-restraint stress-induced gastric ulcer rats,improve gastric tissue pathological injury,and reduce the expression of serum NF-κB p65 mRNA,but there is no significant benefit in reducing the expression of serum TLR4 mRNA and MyD88mRNA.3.Fufang Zhixue Powder can significantly can reverse the dysbiosis of intestinal microbiota in water-immersion and restraint stress-induced ulcer rats,and significantly enriches probiotics such as Lactobacillus、Psychrobacter、Aerococcus、Allobaculum、Faecalitalea.4.Fufang Zhixue Powder significantly upregulates the concentration of glycyrrhizic acid,pyrophosphate,valinomycin,and 10-nitro-oleic acid,and are closely related to the metabolism of phenylalanine,arachidonic acid,arginine,and proline.5.The mechanism of Fufang Zhixue Powder in preventing stress ulcers through"microbiota-metabolite-host"may be related to its ability to the enrichment of probiotics such as Allobaculum and Faecalitalea,promoting the production of short-chain fatty acids,thereby inhibiting oxidative stress and inflammation response. |
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