Effects Of Lactobacillus Plantarum On Intestinal Microbiota Of Precursor Lession In The Early Stage Of Colorectal Cancer Induced By 1,2-Dimethylhydrazine In Rats

Objective:Colorectal Cancer?CRC?is a multi-factor,multi-stage disease involving genetics,environment and lifestyle.As the third most common cancer in humans,annual morbidity and mortality are still rising.In order to reduce the mortality caused by CRC,it is extremely important to find a characteristic lesion that can be used for early detection of CRC.Aberrant Crypt Foci?ACF?is a precancerous lesion that precedes colon adenoma and is the earliest distinguishable morphological feature of CRC.Monitoring changes in ACF can be used to evaluate the results of precursor lession in the early experimental model of colorectal cancer.Intestinal microbes are closely related to colorectal cancer.The former is the latest research hotspot.It has been found that many intestinal bacteria have proinflammatory and carcinogenic properties,and many intestinal bacteria have shown a tendency to inhibit inflammation and cancer development,so it may be against the colon.The occurrence of rectal cancer has a certain impact.Lactobacillus Plantarum?LP?is distributed in a variety of ecological environments and can colonize the gut of humans and other mammals.It catabolizes in the human intestinal tract to produce beneficial small molecular substances,such as Short-chain Fatty Acids?SCFAs?,to promote intestinal health.However,how the bacterium affects the occurrence of CRC by regulating inflammatory mediators,and how to regulate the structure and abundance of intestinal flora,the progression of CRC induced by 1,2-Dimenthylhydrazine Dihydrochloride?DMH?is not yet complete.In this study,the potential mechanism of LP in the treatment of early colorectal cancer was elucidated by exploring the effects of LP on genes related to inflammation-related factors and immune signaling pathways,as well as the differential changes of intestinal microflora abundance and structure under the influence of LP colonization.Methods:Bacterial culture:Lactobacillus plantarum was cultured and identified by MRS medium.Animal experiments:48 8-week-old male Sprague-Dawley Rats?Sprague-Dawley Rats?were randomly divided into 4 groups after 1 week,which were PBS+normal saline control group?group C?,LP+saline control group.Induced tumor group?group L?,PBS+DMH induced tumor group?group DMH?,LP+DMH induced tumor group?group L-DMH?.DMH was injected subcutaneously into the neck of the rat according to different groups,and the concentration was 30 mg/kg rat body weight.On the 7th day after the injection,the cultured LP suspension was administered to the rats in the group once a day(10¹⁰ CFU/ml)for 7 consecutive days.On the 7th day after the end of the gavage,DMH was subcutaneously injected into the back of the neck of the rat,and the concentration was unchanged.On the 7th day after the second DMH injection,the LP suspension was administered again for 7 days,and the concentration was the same as before.On the 7th day after the end of the second gavage,DMH was injected subcutaneously into the neck of the third rat.After that,normal drinking water and diet until the end of the 20th week of the experiment.Rats in the control group were injected with the same dose of normal saline in the back of the neck,and were intragastrically administered with the same dose of PBS solution.The general condition of rats and the abnormal crypt foci of colorectal mucosa were observed Methylthioninium Chloride staining.Cytokine Cyclooxygenase-2?COX-2?,Tumor Necrosis Factor-??TNF-??,Interleukin-8?IL-8?,etc.Detection was performed by Enzyme-Linked ImmunoSorbent Assay?ELISA?.Real-time PCR was used to detect the changes of mRNA expression of Nuclear Factor kappa B?NF-?B?,Signal Transducers and Activators of Transcription 3?STAT3?in each group.Finally,each group of rat feces was collected and analyzed for bacterial colonies by High Throughput Sequencing Technology.Results:LP was successfully cultured and a rat model of precursor lession in the early colorectal cancer was established.The number of ACF in rats:The DMH group was higher than the C group,that is,the modeling was successful.The L-DMH group was lower than the DMH group,and the difference was statistically significant?P<0.05?.The concentrations of serum TNF-?,IL-6,COX-2 and IL-8in the rats were significantly higher than those in the C group?P<0.05?.The expression in the L-DMH group was significantly lower than that in the DMH group.Statistically significant?P<0.05?.The expression of NF-?B and STAT3mRNA in rat colon tissue:NF-?B:There was no significant difference between DMH group and C group,L-DMH group and DMH group,L group and C group?P>0.05?;STAT3:The expression of DMH group was higher than that of C group,and the expression of L-DMH group was lower than that of DMH group?P<0.05?.High-throughput sequencing results showed that the diversity of intestinal flora in DMH group was lower than that in group C.The diversity of intestinal flora in L-DMH group was significantly higher than that in DMH group?P<0.05?.Principal Component Analysis?PCA?found that the main components of the four groups of intestinal flora were different,which was statistically significant?P<0.05?.Community Structure Analysis showed that the levels of major bacteria were compared:Proteobacteria,Actinobacteria,and Firmicutes were higher in the DMH group than in the C group and L-DMH.The content of Bacteroidetes and Spirochaetae in the DMH group was significantly lower than that in the C group and the L-DMH group?P<0.05?.Comparison of genus levels:Escherichia/Shigella,Akkermansia,ClostridiumIV,Eubacterium,Staphylococcus,in the DMH group.The number of L-DMH group increased,which was statistically significant?P<0.05?.The number of Blautia,Provotella,Lactobacillus was higher than that of C group and The L-DMH group decreased,which was statistically significant?P<0.05?.Conclusions:LP can reduce the formation of abnormal crypt foci in early stage of colorectal cancer,inhibit the expression of inflammatory factors COX-2,IL-6,TNF-?,IL-8 and STAT3 genes,and improve the intestinal flora caused by DMH.The imbalance between degree and structure promotes the production of beneficial metabolites by bacteria,thereby reducing the risk of colorectal cancer.