The Regulatory Effects And Mechanisms Of LDR-induced Gut Microbiota Changes On Type 2 Diabetes

Type 2 diabetes(T2DM)is a metabolic disease characterized by rising blood sugar and insulin resistance.Its complications include chronic kidney disease,cardiovascular disease,ketoacidosis,retinopathy and neurodegenerative changes.In developing and developed countries,the death rate of type 2 diabetes and its complications is rising due to the change of modern people’s lifestyle,the increase of high energy and high calorie diet,long-term sitting and lack of exercise.At present,the main treatment of T2DM is to control blood sugar through drug therapy and insulin injection.New methods and strategies for T2DM are also being explored.There are 10 trillion bacteria in the human gut,with a total number of genes about150 times the number of human genes.They are also known as the"second genome"of the human body.They can affect people’s nutrient absorption,energy metabolism,and immune function.More and more evidence shows that the occurrence and development of T2DM is also closely related to these intestinal flora,and T2DM can cause disorders of intestinal flora,The disorder of intestinal flora leads to metabolic abnormalities,chronic systemic inflammation,insulin resistance,etc.,making the symptoms of T2DM more severe,forming a vicious cycle.This study used high-fat diet and low-dose streptozotocin(STZ)injection to induce T2DM in mice,and then explored the effect and mechanism of low-dose ionizing radiation(LDR)on intestinal flora and metabolites in normal mice and type 2diabetes mice,aiming to change the disorder of intestinal flora in type 2 diabetes mice,alleviate its inflammatory response,and reverse the changes of its metabolites,To provide a new idea for clinical relief of T2DM symptoms.Objective:To explore the changes of intestinal flora and metabolites in type 2 diabetes mice induced by low-dose radiation,and the alleviating effect of low-dose radiation on T2DM induced inflammatory response and its mechanism,so as to provide a theoretical basis for the clinical application of low-dose radiation.Method:1.C57BL/6 mice were fed a high fat diet for 16 weeks and injected with STZ solution at a concentration of 30 mg/kg.The success of the T2DM model was determined by measuring body weight,glucose tolerance,and blood glucose levels.After successfully establishing the model,the experiment was divided into Con group,LDR group,T2DM group,and T2DM+LDR group.The irradiation dose was 75 m Gy,with a dose rate of 0.0134 Gy/min,and was irradiated every other day for a total of 14times over four weeks,with a total dose of 1.05 Gy.2.Use a blood glucose meter to detect blood glucose values of mice in each group,and monitor weight changes.After the experiment was completed,the mice were anesthetized and killed,and the weight of each organ was weighed.3.Collect fecal and serum samples from each group of mice,and send them to Beijing Biotech Co.,Ltd.for 16S r DNA fragment amplification and high-throughput sequencing,as well as serum non-target metabolomics analysis.4.The pathological changes of intestinal tract,pancreas and testis were observed by HE staining.While Immunohistochemistry(IHC)was used to detect protein expressions of TLR4,My D88,NF-κB p65,p NF-κB p65 and p38 from the mouse intestine,pancreas and testis,and use Image J software for quantitative analysis.5.Western Blot was used to detect the expression of TLR4,My D88,NF-κB p65and p NF-κB p65 in mouse testis.6.The m RNA expressions of My D88,NF-κB p65,TNF-αand IL-1 in intestinal and testicular tissues of mice in each group were detected by quantitative real-time PCR(qRT-PCR).7.Statistical analysis:SPSS 24.0 software was used for analysis.The experimental data obtained were expressed as mean±standard error(~_x±s).The comparison between the two groups was conducted using independent sample t-test,with P<0.05defined as significant difference.Result:1.Successful establishment of mouse type 2 diabetes modelAfter 16 weeks of high fat diet feeding,the mice showed abnormal glucose tolerance,and were subsequently injected with STZ solution.After three days,the blood glucose was higher than 11.1 mmol/L,significantly higher than that of the control group(P<0.05),indicating that the mouse T2DM model was successfully established.2.LDR can regulate the damage of type 2 diabetes miceAfter irradiation,the blood sugar and body weight of type 2 diabetes mice decreased significantly compared with those of non irradiated mice.This indicates that low dose irradiation can affect changes in body weight and blood sugar,make body weight tend to normal in mice,and weaken insulin resistance.The HE results showed that there was a certain degree of destruction of the intestinal villus structure in the T2DM group,with severe destruction of the upper villus.The outer membrane of the T2DM group and the T2DM+LDR group became thinner,but there was no significant destruction of the upper villus in the T2DM+LDR group.Pancreatic T2DM group has severe intra pancreatic congestionβCell morphology is incomplete,but after LDR irradiation,there is a certain degree of repair.In the reproductive system of T2DM group,interstitial looseness of seminiferous tubules and thickening of blood vessel walls can be seen.After irradiation,the blood vessel walls have recovered.3.LDR can change the composition of intestinal flora in type 2 diabetes miceThe results of 16S r DNA fragment amplification and high throughput sequencing showed that there were changes in the composition of the flora at the level of phyla,class,order,family,genus,and species.Specifically,Desulfobacterota increased in the T2DM group,with a statistical difference(P<0.05).Compared with the T2DM group,Desulfobacterota in the T2DM+LDR group had no statistical difference,but showed a downward trend;Verrucomicrobiota and Proteobacteria decreased significantly in the T2DM group,with a statistically significant difference(P<0.05).Proteobacteria showed an upward trend in the T2DM+LDR group,but there was no statistically significant difference;compared with Con group,LDR group had no significant changes in the above flora.4.LDR can change the richness and diversity of intestinal flora in type 2diabetes mice.According to alpha diversity and beta diversity analysis,LDR will change the abundance and diversity of intestinal flora in type 2 diabetes mice to that in normal mice.5.Effect of LDR on metabonomics of type 2 diabetes miceSerum metabolomic analysis showed that compared with the control group,the energy metabolism and lipid metabolism of mice in the T2DM group changed,and the above changes could be changed in the LDR+T2DM group,specifically manifested as D-(+)-raffinose pentahydrate,Chrysin 7-glucuronide,Nepetaside,Serratin,MG(0:0/20:4(5Z,8Z,11Z,14Z)/0:0)and 2,3-octadienylglycine were downregulated in the T2DM group and upregulated in the T2DM+LDR group.6.Effect of LDR on TLR4/My D88/NF-κB pathway protein expression in pancreas and testis of type 2 diabetic miceIHC results showed that the expressions of TLR4,My D88 and NF-κB p65 in intestinal tissue,pancreas and testis in T2DM group were increased compared with Con group,and the expressions of p NF-κB p65 and p38 in intestinal tissue were increased compared with Con group.However,the expressions of TLR4,My D88 and NF-κB p65in intestinal tissue,pancreas and testis of LDR+T2DM group were decreased compared with that of T2DM group,and the expressions of p NF-κB p65 and p38 in intestinal tissue were also decreased compared with that of T2DM group.After quantitative treatment,statistical analysis was performed,P<0.05.Western Blot showed that TLR4,My D88,NF-κB p65 and p NF-κB p65 in testicular tissue of T2DM mice increased compared with Con group.The protein expressions of TLR4,My D88,NF-κB p65 and p NF-κB p65 in testicular tissue of T2DM mice were decreased after LDR.It is suggested that diabetes can activate this pathway and participate in inflammatory response,but LDR can reduce the expression of inflammatory proteins in type 2diabetes mice and reduce the inflammatory effect of diabetes.7.Effect of LDR on TLR4/My D88/NF-κB pathway inflammatory factor gene expression in intestinal and testis of type 2 diabetic miceqRT-PCR results showed that:The levels of TLR4,My D88,NF-κB p65,TNF-αand IL-1 in intestinal tract and testis of T2DM group were significantly increased compared with those of control group,but after LDR,the levels of TLR4,NF-κB p65and My D88 in intestinal tract of normal mice were significantly increased after irradiation.TNF-αand IL-1 were increased,and the NF-κB p65 and TNF-αgenes in testis were also increased,indicating that LDR can increase the expression of inflammatory factors in the gut and testis of normal mice,but can also decrease the expression of inflammatory factors in the gut and testis of type 2 diabetes mice.Conclusions:1.In this study,high-fat diet and STZ injection were used to successfully construct a mouse model of type 2 diabetes,which could be used for subsequent experiments;2.Low dose radiation can reduce the blood sugar and body weight of diabetic mice,and alleviate the damage of pancreas,small intestine and testis;3.The intestinal flora of type 2 diabetic mice was changed,and the species richness and diversity of the mice were decreased compared with that of normal mice.The species richness and diversity of type 2 diabetic mice were normalized by LDR irradiation.4.In mice with type 2 diabetes,the desulfurizing vibrio bacteria increased,while LDR could alleviate this phenomenon;5.As gram-negative bacteria,lipopolysis,the component of the outer wall of Vibrio vulcanis cell wall,releases inflammatory cytokines through TLR4 signaling pathway,causing chronic intestinal inflammation,weakening insulin signal and producing insulin resistance,while LDR can reduce chronic intestinal inflammation and insulin resistance.6.Non-target metabolomics showed that amino acid metabolism,energy metabolism and lipid metabolism were abnormal in type 2 diabetic mice,and LDR could alleviate the damage in type 2 diabetic mice to some extent.