Function Study Of Nprl2 Gene In Drosophila Gastrointestinal Aging Model

Aging occurs in all living organisms and is accompanied by tissue degradation that produces a variety of age-related diseases.It was discovered that the inhibitor of target of rapamycin complex 1(TORC1),a conserved complex that mediates nutrient status and cell metabolism,can extend an individual's lifespan and inhibit age-related diseases in many model organisms.Here,we use a strain of the inhibitory gene nprl2 of TORC1(Nprl2 protein is one component of the GATOR1 complex),to investigate the effect of increased TORC1 activity on gastrointestinal tract(GI)digestive dysfunction in Drosophila.we found that the lifespan of nprl2 mutant drosophila was significantly reduced compared to that of control.Statistics show that with age,the crop of the nprl2 mutant(it function similar to the stomach of a mammal)increased significantly and there was a phenomenon of food accumulation.In order to clarify the function of Nprl2 in GI.male Drosophila was taken as the experimental object to study the regulation of nprl2 gene on food transport and intestinal aging in crop.The main results are as follows:(1)By staining the contours of crop cells by immunofluorescence,it was found that the increase in the crop of nprl2 mutant was caused by the accumulation and expansion of food and the expansion of the membrane.After feeding the blue dye food to the flies,it is found that food stays longer in nprl2 mutants,and the distribution of bright blue food was determined by UV spectrophotometer,the blue dye food is more distributed in the crop(P<0.01)and less in the gut(P<0.001).Therefore,after the nprl2 gene mutation,food accumulation occurs in the crop,and the rate of food digestion slows down..(2)The results show that there is no significant difference in food intake in Drosophila by CAFE method.However,the Defecation rate of Drosophila with nprl2 mutant was significantly reduced(P<0.001),and the shrinkage of the mutant Drosophila crop was significantly slower(P<0.001).The loss of gastric motility in nprl2 mutants may be responsible for the increase of crop and food accumulation in nprl2 mutant.(3)By measuring the drosophila gut,it was found that the nprl2 mutant had a shorter and thiner gut(P<0.05,P<0.001),it is considered that the intestinal homeostasis was out of balance.By measuring the related expression of intestinal epithelial cell stem cells,it was found that the nprl2 mutant Drosophila ISCs overproliferated(P<0.001),and the differentiated intestinal endocrine cells(EEs)were significantly reduced(P<0.05).Then the intestinal lipid storage was determined by Nile red staining,the results showed that the intestinal lipid storage of nprl2 mutant Drosophila was significantly reduced(P<0.001).Intestinal damage agent SDS was fed to drosophila,and the lifespan of nprl2 mutant Drosophila was significantly reduced(P<0.001).Finally,the expression of intestinal antimicrobial peptide genes were measured,and the expression of nprl2 mutant Drosophila antimicrobial peptide was found to be significantly increased(P<0.001).These phenomena suggest that the gut of the nprl2 mutant Drosophila is accelerating aging.(4)The above experiments were carried out by using nprl2;TorA948V Drosophila.It was found that when the activity of TORC1 decreased,the crop became smaller,the metabolism of food increased,and the distribution of food in the diet decreased to the wild-type state of the control group,and the contraction rate of the crop also return to the wild-type state.Therefore,it is inferred that the increase of TORC1 activity in the nprl2 mutant is the cause of age-related GI dysfunction.(5)Drosophila was fed with TORC1 inhibitor rapamycin.It was found that after treat with rapamycin,the phenomenon of food accumulation in crop was recovered obviously,It indicated that rapamycin can inhibit GI disorder caused by nprl2 deficiency.It further indicated that nprl2 protein maintains normal GI function by inhibiting TORC1 activity.Our research results show that nprl2 mutant Drosophila GI shows the characteristics of aging Drosophila,which is due to the high activity of TORC1,suggesting that excessive increase of TORC1 activity may accelerate the aging of GI digestive system...