Effect Of ERAS On Intestinal Flora And Oxidative Stress In Patients Undergoing Colorectal Cancer Surgery

【Objectives】In this study,the clinical indexes,intestinal microbial composition and oxidative stress response of patients undergoing elective colorectal cancer（CRC）surgery under the traditional operation mode and the enhanced recovery after surgery（ERAS）mode were analyzed and compared,in order to explore the differences and possible mechanisms of postoperative rehabilitation for patients undergoing elective CRC surgery under the two modes.【Methods】1.The patients who were diagnosed as CRC by colorectal surgery in Yunnan Tumor Hospital from September 2021 to March 2022 were selected as the research objects.Each participant has been told to sign the informed consent form.The patients were divided into traditional group（Tra-group）and ERAS group according to the difference of perioperative treatment.The research has been approved by the Ethics Committee of Yunnan Cancer Hospital.2.The postoperative indexes of each patient were recorded to determine the accelerated rehabilitation effect of ERAS on patients with CRC after operation.（1）The time of first anal exhaust and defecation was recorded for comparing the recovery of gastrointestinal function between the two groups.（2）Inflammation and nutritional indicators:About 5m L of venous blood was taken from the patients before operation and on the 1st and 3rd day after operation respectively to detect the indexes of PCT,CRP and ALB.（3）Record the length of stay of patients after surgery and observe whether complications occurred during the postoperative hospitalization.3.The expression levels of inflammatory factors IL-1βand TNF-αin intestinal tissues of patients were determined by ELISA.The expression of the intestinal tight junction proteins Occludin and Claudin were detected by western blotting.4.16S rRNA high-throughput sequencing was used to analyze the intestinal microflora of patients in the traditional group and ERAS group before and after operation.To understand the effects of the two modes on the intestinal microbial composition of patients undergoing CRC surgery and to screen the dominant and specific microflora in the ERAS mode.5.ELISA and qPCR were respectively used to evaluate the expression of oxidative stress related factors MDA,ROS,and antioxidant stress related factors GSH,SOD in intestinal tissue.6.Iron metabolism in intestinal tissue was detected by the relative assay kits.WB and IHC were used for detecting biomarker proteins of ferropotosis in intestinal tissues,such as ACSL4,GPX4,FTH and HO-1.【Results】1.A total of 56 newly diagnosed CRC patients were included in our hospital,including 28 cases in the traditional group and 28 cases in the ERAS group.All patients received laparoscopic radical resection of colorectal cancer.There was no significant difference in age,gender composition,BMI,tumor location and clinical stage between the two groups（P>0.05）.2.The statistical results showed that the recovery time of intestinal function,pain score,nutritional and inflammatory indexes ALB,PCT,CRP and hospitalization time of patients in ERAS group were better than those in traditional group（P<0.01）.3.The expression levels of pro-inflammatory cytokines IL-1β（P<0.01）and TNF-α（P<0.001）in intestinal tissue of patients with CRC undergoing radical resection in ERAS group were significantly lower than those in traditional group.However,the expression of tight junction proteins Occludin（P<0.05）and Claudin（P<0.0001）was significantly higher than that of the traditional group.4.16S rRNA high-throughput sequencing was performed on the preoperative and postoperative fecal samples of 10 random patients from traditional group and era group.The results showed that there was no significant difference in the richness,diversity and structure of intestinal flora before operation between the two groups（P>0.05）,as well as the abundance of main groups at the level of phylum and genus（P>0.05）.There was no significant difference in abundance and diversity of intestinal flora and abundance of major groups at phylum level between the two groups of patients after operation（P>0.05）.The main differences are at the genus level,the abundance of Lachnospiraceae＿UCG＿010,Prevotella＿2,Anaerotruncus,Fusicatenibacter,Catenibacterium,Eubacterium＿coprostanoligenes＿group,Candidatus＿Soleaferrea,Leuconostoc,Microtrichales,Ilumatobacteraceae,CL500＿29＿marine＿group were higher in ERAS group（P<0.05）.And the abundance of Eubacterium＿saphenum＿group,Megamonas,Sutterella,Synergistetes,Clostridiales＿vadin BB60＿group,S5＿A14a,Plesiomonas were higher in the traditional group（P<0.05）.The functional analysis of Tax4Fun indicated that"Carbohydrate Metabolism","Amino Acid Metabolism","Metabolism of Cofactors and Vitamins","Glycan Biosynthesis and Metabolism"categories related to energy metabolism significantly increased in intestinal flora of patients in ERAS group.5.The results of ELISA showed that the contents of ROS（P<0.001）and MDA（P<0.01）in intestinal tissue of ERAS group were significantly lower than those of traditional group,while GSH activity was significantly higher than that of traditional group（P<0.001）.The results of qPCR showed that the levels of SOD m RNA（P<0.05）and GSH m RNA（P<0.01）in intestinal tissue of ERAS group were significantly higher than those of traditional group.The results indicate that ERAS not only down-regulates the production of ROS and MDA,but also up-regulates the production of GSH and SOD.6.The levels of Iron（P<0.05）and Fe ²⁺（P<0.0001）in intestinal tissue of ERAS group were significantly lower than those of traditional group.The results of qPCR showed that the ferropotosis pathway biomaker GPX4 m RNA in ERAS group was significantly up-regulated（P<0.01）as compared with the traditional group,while HO-1 m RNA（P<0.01）and TF m RNA（P<0.05）were significantly down-regulated;WB results showed that the expression levels of GPX4 and FTH in ERAS group were significantly higher than those in traditional group（P<0.05）,while the relative expression levels of ACSL4（P<0.05）and HO-1（P<0.001）were significantly decreased.The IHC results are consistent with the above results.ERAS inhibited the development of ferropotosis.【Conclusions】1.ERAS can promote the recovery of intestinal function in patients with CRC after operation and shorten the hospitalization time after operation.2.Postoperative serum inflammatory indexes of PCT and CRP in ERAS group were lower than those in traditional group.3.ERAS exerts anti-inflammatory effect by inhibiting local inflammatory mediators IL-1βand TNF-α,and can reduce the damage of tight junction proteins Occludin and Claudin in the intestinal mucosal barrier.4.ERAS can protect the destruction of intestinal microbial homeostasis and up-regulate the abundance of probiotics.ERAS may promote intestinal functional recovery and enhance intestinal barrier by regulating intestinal microorganisms.5.ERAS can reduce the body’s oxidative stress reaction and inhibit the development of ferropotosis.