The Adjuvant Treatment And Molecular Mechanism Of Probiotic Compounds In Patients With Gastric Cancer After Gastrectomy

Background and objective:Gastric cancer is one of the most common malignant tumors in the upper digestive tract in the world,accounting for the third leading cause of cancer-related death and the second most common malignant tumor in China.Comprehensive treatments,mainly radical surgery,are the main mode of gastric cancer treatment currently.Despite the comprehensive treatment adopted,the curative effect is still not satisfactory.Surgery leads to destruction of normal physiological structure of digestive tract,imbalance of intestinal microecology,impairment of intestinal mucosal barrier and immunity,aggravation of intestinal inflammation,which seriously affects the recovery and prognosis of patients during perioperative period.Intestinal microecology refers to various complex microbes residing in the intestinal tract.Studies have found that intestinal microecology balance is closely related to intestinal inflammation,immunity and intestinal mucosal barrier.Probiotics refer to microbes that exert beneficial effects on the host after being injected into the body and are important members of intestinal flora.Our previous studies have found that probiotic drugs can promote the recovery of gastric cancer patients after gastrectomy by adjusting the intestinal microbial composition,reducing inflammation and enhancing immunity.However,considering the existing probiotic drugs contain"old bacteria"and strains that pose a potential threat to human health,we isolated and screened bacteria from the feces of healthy volunteers in Wuyuan"cancer-free village"and obtain 3 probiotics(Lactobacillus plantarum,L.rhamnosus and L.acidophilus),compounded with Bama"Changshou village"probiotics(Bifidobacterium animalis),and conducted in vitro probiotic evaluation.Finally,these4 probiotics were synthesized into a probiotic compound to explore its therapeutic effect and mechanism of action on gastric cancer recovery after gastrectomy,and provide new ideas for clinical administration.Methods:1.SD rat model1)Establishment of gastric precancerous lesion model in SD rats:male SD rats aged 6-8 weeks were divided into group C(control group,n=12)and PLGC group(gastric precancerous lesion model group,n=48).Group C was treated with pure water free drink,normal feed,and 2 mL distilled water was infused into the stomach every other day for comparison.In PLGC group,0.15 g/L MNNG solution was used for free drinking;2 mL of 2%salicylic acid and 0.5 ml of 15%NaCl at 55?were given to the stomach every other day,and then combined with starvation and satiety disorder to create a model(normal feeding for 2 days and fasting for 1 day);2)Establishment of gastrectomy model:After PLGC model is successfully established,the gastrectomy model were stablished,and type I gastrectomy was used.When the model is established,groups were divided into C group(control group,n=10),M group(model group,n=12),PL group(low-dose probiotic compounds administration group;Lactobacillus plantarum 10~7 CFU/mL,L.rhamnosus 10~7CFU/mL,L.acidophilus 10~7 CFU/mL,Bifidobacterium animalis 10~7 CFU/mL;n=12)and PH group(high-dose probiotic compounds administration group;Lactobacillus plantarum 10~9 CFU/mL,L.rhamnosus 10~9 CFU/mL,L.acidophilus 10~9 CFU/mL,Bifidobacterium animalis 10~9 CFU/mL;n=12)and treated according to groups.3)Effect and mechanism of probiotic compounds:Western-blot was used to detect the expression of intestinal inflammation-related proteins(TLR4,MyD88,NF-?B and p-NF-?B)and intestinal mucosal barrier proteins(MLCK,MLC and p-MLC)in rats.ELISA was used to detect the expression of serum inflammatory factors(IL-1?,IL-6 and TNF-?)in rats.Blood cell count was used to detect the percentage of rat blood cells(white blood cells,lymphocytes and central granulocytes),and HE staining was used to evaluate the damage of rat gastric tissues and intestinal mucosae,immunohistochemistry and immunofluorescence were used to detect the expression of rat intestinal mucosal immune cells(CD3~+T,CD4~+T and CD8~+T)and intestinal epithelial tight junction proteins(ZO-1,Claudin-1 and Occludin),and high-throughput sequencing was used to analyze the difference of intestinal microbial composition of rats in each group.2.Clinical verification1)From October 2018 to July 2019,100 gastric cancer patients in the Second Affiliated Hospital of Nanchang University were included according to requirements.They were randomly divided into AC group(placebo treatment group after gastrectomy)and ACP group(probiotic compounds treatment group after gastrectomy).Placebo or probiotic compounds were respectively taken for 5-7 days after gastrectomy.2)Collect blood samples of gastric cancer patients before and after gastrectomy,and count blood cells(white blood cells,lymphocytes and central granulocytes),and analyze the differences between groups by statistics to evaluate the effects of probiotic compounds on the inflammation and immunity of patients after gastrectomy;3)Collect feces samples of gastric cancer patients before and after gastrectomy,and analyze the difference of intestinal microbial composition in each group by high-throughput sequencing.Results:1.SD rat model1)High-dose probiotic compounds had the best therapeutic effect on promoting postoperative recovery of gastrectomy rats.2)The probiotic compounds reduced the expression level of intestinal inflammatory pathway proteins(TLR4,MyD88 and p-NF-?B),reduced the count of blood inflammatory cells(leukocytes,central granulocytes),and reduced the expression level of serum proinflammatory factors(IL-1?,IL-6 and TNF-?);3)The probiotic compounds increased the proportion of blood immune cells(percentage of Lymphocytes)in rats,regulated the number of intestinal mucosal immune cells,increased the number of CD4~+T,decreased the number of CD8~+T,increased the CD4/CD8 ratio,and restored the intestinal mucosal immunity;4)The probiotic compounds inhibited activation of intestinal mucosal barrier transduction pathway MLCK/p-MLC,inhibited degradation of intercellular tight junction proteins(ZO-1,Claudin-1 and Occludin),and further plays a role in protecting host intestinal mucosal barrier;5)The probiotic combinations increased the relative abundance of beneficial intestinal bacteria Akkermansia,Bacteroides and Parabacillides,and reduced the relative abundance of pathogenic bacteria Clostridium,Proteus and Bilophila,and stabilize the intestinal microbial balance.2.Clinical verification1)The probiotic combinations reduced blood inflammatory cells(white blood cells,central granulocytes)of patients after gastrectomy,and increases blood immune cells(percentage of lymphocytes)of patients;2)The probiotic combinations increased the relative abundance of Lactobacillales,Bacteroides and Phascolarctobacterium,and decreased the relative abundance of pathogenic bacteria Dorea and Pseudomonas,and maintains the stability of intestinal microbe balance.Conclusion:1)Probiotic compounds can promote the recovery of gastric cancer patients after gastrectomy.2)The probiotic compounds supplement promotes the formation of new microecological balance in intestinal microbe by increasing the relative abundance of beneficial bacteria and reducing the relative abundance of pathogenic bacteria;Stable tight junction protein structure between intestinal epithelium and maintenance of intestinal mucosal barrier function;Stabilize the number of intestinal mucosal immune cells and maintain the intestinal mucosal immune ability;Downregulation of inflammatory signaling pathway,prevention of release of pro-inflammatory factors,reduction of body and intestinal inflammatory response,and ultimately promoting postoperative recovery and possibly improving the prognosis of patients.