Improvement And Mechanism Of Engineered Clostridium Butyricum-pMTL007-GLP-1 On Type 2 Diabetes In Mice

Background and aimsDiabetes mellitus is a multifactorial metabolic disease characterized by elevated blood glucose,and its prevalence has increased exponentially in recent years.According to the World Health Organization,diabetes will be the seventh leading cause of death worldwide by 2030.Glucagon-like peptide-1(GLP-1)is an intestinal hormone with glucose-concentration-dependent hypoglycemic effects,but because its plasma half-life is less than 2 minutes,most products must be administered continuously by intravenous drip to produce efficacy.Although GLP-1 analogs and Dipeptidyl peptidaseⅣ(DPP-IV)inhibitors have been developed to overcome this deficiency and some progress has been made,a series of problems such as the high cost of the drugs and the demand for long-term injection still plague the affected population.Notably,the emergence of genetically engineered bacteria has broken this stalemate.By integrating the target gene into a specific site on the bacterial chromosome and delivering it to the human intestine for action by oral administration,this active biologic drug has yielded breakthroughs in various disease areas.Therefore,in this project,Clostridium butyricum(C.butyricum)was used as drug carrier to construct an engineered bacterium that can sustainably express GLP-1,while its efficacy was evaluated through type 2 diabetes mellitus(T2DM)mice model,with a view to providing data support for the research and development of diabetes treatment drugs with low side effects,low economic cost,and can be administered orally.Experimental methods1.Construction of engineered bacteria and in vitro property evaluationThe recombinant plasmid p MTL007-GLP-1 was integrated into the C.butyricum genome by bacterial conjugation to construct C.butyricum-p MTL007-GLP-1engineered bacteria,and the expression of GLP-1 in its crushed supernatant was detected by ELISA.Seven short-chain fatty acids in bacterial culture supernatants,namely acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid and hexanoic acid,were determined by targeted metabolomics.The absorbance(at 600 nm)of the bacteria was monitored by turbidimetric method for 24h and the growth curve was plotted.The resistance of the bacteria was evaluated by acid and bile salt tolerance tests.2.Efficacy evaluation of engineered bacteria on T2DM mice and mechanism investigation1)T2DM mice model establishment and evaluation:Eight-week-old C57BL/6male experimental mice were purchased,and 12 were selected as the normal group(C group).The remaining mice were fed with high-fat diet(HFD)combined with streptozotocin(STZ)intraperitoneally for T2DM modeling.Mice were included in the study when their random blood glucose was above 16.7 mmol/L and remained stable.The enrolled mice were randomly divided into four groups of 12 each:model group(M group),wild-type C.butyricum group(Cb group),exenatide group(E group),and C.butyricum-p MTL007-GLP-1 engineered bacteria group(Cb-GLP-1 group).The C and M groups were intragastric administrated with sterile saline daily,the same operation was performed on the Cb and Cb-GLP-1 groups with 10~8CFU/m L of corresponding bacterial solution,while E group was injected intraperitoneally with 24nmol/kg exenatide daily.During the 10 weeks of treatment,the mice were monitored for blood glucose and body weight changes every other week,with oral glucose tolerance test and insulin tolerance test being performed two weeks before the end of the experiment for efficacy evaluation.2)Mechanism analysis:Liver function impairment and lipid deposition was accessed by lipid level(TC,TG,LDL-C,HDL-C)testing,liver function index(AST,ALT)testing and oil red O staining.The expression of GLP-1 in pancreatic tissue was detected by ELISA.Expression levels of key pancreatic apoptotic pathway proteins(p-PI3K,PI3K,p-AKT,AKT,Bax,Bcl-2,Caspase-3,cleaved-Caspase-3),key insulin secretory pathway proteins(GLP-1R,AC,PKA,PDX-1)and colonic tight junction proteins(Occludin,ZO-1)were detected by Western blot.Morphological and structural damage of pancreatic tissue was observed by H&E staining.Pancreatic insulin secretion was analyzed by immunofluorescence.Transcript levels of colonic inflammatory factors(TNF-α,IL-6,IL-1β)was monitored by q PCR.The effects of different experimental treatments on the number and species of intestinal flora in mice were analyzed by high-throughput sequencing.Experimental results1.In vitro evaluation on the properties of engineered bacteria(1)ELISA detected the expression of GLP-1 in the crushed supernatant of the engineered bacteria,indicating the successful construction of the engineered bacteria;(2)There was no difference in short-chain fatty acid secretion between the engineered and natural bacteria,which indicated that the construction process of the engineered bacteria had no effect on the short-chain fatty acid secretion performance of C.butyricum;(3)The engineered bacteria entered the stabilization phase after 16 h of incubation,and no significant difference has been found from the growth of wild-type C.butyricum;(4)The engineered bacteria exhibited excellent acid and bile salt tolerance.2.Efficacy evaluation of engineered bacteria on T2DM mice and mechanism investigation(1)The Cb-GLP-1 group significantly reduced blood glucose,alleviated weight loss,and improved glucose tolerance and insulin tolerance in mice compared to the M group;(2)In group M,the levels of TG,TC and LDL-C were increased and HDL-C level was decreased,accompanied by some hepatic impairment(increased AST and ALT levels)and liver lipid deposition,which was reversed by treatment in Cb-GLP-1group;(3)Compared with islet morphological lesions in pancreatic tissue of the M group,the Cb-GLP-1 group promoted islet morphological recovery,while slowing down the process of islet apoptosis by upregulating the expression levels of p-PI3K/PI3K and p-AKT/AKT,inhibiting the ratio of downstream Bax/Bcl-2 and the expression of cleaved-Caspase-3/Caspase-3;(4)Compared with the M group,the Cb-GLP-1 group promoted pancreatic GLP-1 expression,which in turn upregulated the expression of insulin secretion-related proteins(GLP-1R,AC,PKA,PDX-1)to promote insulin secretion;(5)In contrast to the M group that existed decreased intestinal barrier function and inflammatory infiltration,the Cb-GLP-1 group enhanced the expression of mouse colonic tight junction protein(Occludin,ZO-1)and significantly inhibited the transcriptional levels of TNF-α,IL-6 and IL-1β(p<0.01);(6)High-throughput sequencing of intestinal microbiome showed that the microbial diversity of the Cb-GLP-1 group was increased and its microbial composition was closer to normal level compared to the M group.ConclusionsIn conclusion,the C.butyricum-p MTL007-GLP-1(Cb-GLP-1)constructed in this study could express GLP-1 normally and perform well in growth,secretion and resistance.In the T2DM mice model,Cb-GLP-1 could regulate dyslipidemia and ameliorate liver function impairment,inhibit islet cell apoptosis and promote insulin secretion to lower blood glucose and relieve weight loss,and suppress colonic inflammation by regulating intestinal flora homeostasis.