Probiotics And Prebiotics Improve Dry Eye In Type 2 Diabetic Mice Through The Gut-Eye Axis

Background: China has the largest number of patients with diabetes mellitus(DM)across the world,mainly with type 2 diabetes mellitus(T2DM).DM has become one of the major causes of ocular surface diseases which are attracting extensive attention because they can affect the quality of life of those related patients.Previous studies have shown that intestinal flora dysbiosis exists in DM patients,and there is a relationship between intestinal flora dysbiosis and the development of ocular diseases,thus regulating intestinal flora can be a novel target for ameliorating ocular diseases.Clinically,probiotics and prebiotics have been used for the treatment of intestinal diseases,but whether probiotics and prebiotics can improve the dry eye of type 2 diabetic mice by regulating intestinal flora through the gut-eye axis remain to be illuminated.Objective: To establish a mouse model of T2 DM and observe the effects of probiotics and prebiotics on dry eye symptoms in T2 DM mice;to detect the barrier function of the intestinal tract and ocular surface,level of oxidative stress,apoptosis,and inflammation of the body.To explore the possible mechanisms of the effects of probiotics and prebiotics preliminarily.To conduct a combined analysis of intestinal microbial diversity and non-targeted metabolomics in mice which may suggest the possible sites of probiotics and prebiotics to alleviate the symptoms of diabetic dry eye.We also conducted a combined analysis of intestinal microbial diversity and non-targeted metabolomics in mice to identify possible loci of action of probiotics and prebiotics in alleviating dry eye symptoms in diabetes.These studies will provide a theoretical and experimental basis for the use of probiotics and prebiotics in the prevention and treatment of diabetic dry eye.Methods: A high-fat diet was used to induce obesity in mice,followed by continuous low-dose intraperitoneal injection of streptozotocin to establish a T2 DM mouse model.Body weight,blood glucose,and tear secretion were examined,and pathological changes of the cornea,lid gland,lacrimal gland,conjunctiva,and intestine were observed by staining.The T2 DM mice were randomly divided into the model(saline)group,probiotic group,and prebiotic group.Then they were gavaged for 6 weeks continuously,with the daily food intake and weekly body weight recorded.Tear volume was detected after treatment.The corneal epithelial injury model was established,and the healing of corneal wounds in each group was observed at different times.Blood,fecal,ocular,intestinal,and liver samples were collected,and the visceral fat was weighed.Then,histopathological changes were examined by observing the above samples.Apoptosis was detected by applying TUNEL staining,and the expression of relevant proteins was detected by immunohistochemistry,immunofluorescence staining and western blot.The relative expression of mRNA of related genes was detected by RTPCR,serum oxidase and antioxidant enzymes was detected by the automatic biochemical analyzer,and serum cytokine levels were detected by ELISA.One-way ANOVA and Tukey intergroup multiple comparison tests were performed on the data of each group using Graph Pad Prism software,and P<0.05 was considered statistically significant.16 S r RNA gene sequencing was used to detect changes in fecal microbiota in each group of mice,and correlation analysis between microbiota and serum indicators and dry eye indicators was performed using Spearman’s method.Non-targeted metabolomics was applied to detect changes in the fecal metabolic profile of each group of mice,and correlation analysis was performed between the differential genera and metabolites.Results:(1)Compared to controls,T2 DM mice had increased body weight,increased blood glucose,and decreased lacrimal gland secretion,accompanied by more sodium fluorescein staining of the cornea.Pathological staining showed an increase in the volume of the alveoli in the lid and lacrimal glands of T2 DM mice,more lipid deposition in the lid glands,and a decrease in the number of cup cells secreting glycogen and other substances in the conjunctiva and intestine.(2)Compared with the model group,probiotics and prebiotics had no significant effects on mice’s body weight,food intake,and visceral fat weight.Prebiotics significantly reduced the content of glycated serum proteins in the serum of T2 DM mice,and probiotics significantly reduced the content of triglycerides in the serum of T2 DM mice.Both probiotics and prebiotics increased tear secretion and improved corneal sodium fluorescein staining in T2 DM mice.The arrangement of conjunctival goblet cells in the DM group was disordered and the number was reduced,while probiotics and prebiotics increased the number of conjunctival goblet cells.Neither probiotics nor prebiotics could promote the healing of corneal epithelial wounds.(3)Compared with the model group,the application of probiotics and prebiotics brought many changes: increased expression of tight junction proteins such as ZO-1 or Occludin;increased ki67-positive cells in the conjunctiva,lacrimal gland,and colon whereas decreased apoptotic cells in cornea and colon;increased content of SOD whereas decreased MDA in serum;decreased infiltration of immune cells as well as the expression of inflammatory factors in colon,serum and lacrimal gland.Mechanically,the relative mRNA expression of TLR4,MyD88,and NF-κB,together with the protein expression of p-NF-κB and p-Akt,were significantly increased in the colon of T2 DM mice compared with the control group,while probiotics and prebiotics treatment can significantly decrease these changes in T2 DM mice.In addition,prebiotics can also significantly reduce TLR4 protein expression in T2 DM mice.(4)Compared with the control group,the abundance of Muribaculaceae,Akkermansia and Lactobacillus communities in the intestine of the T2 DM mice was significantly reduced,the structure of the microbiota was significantly altered,where the proportion of the phylum Bacillus was reduced,and the proportions of Actinobacteriota,Bifidobacterium,Ileibacterium,Akkermansia and Lachnospiraceae was increased.Compared with the T2 DM group,probiotics and prebiotics had a significant effect on the structure of the intestinal flora.It increased the abundance of Bifidobacterium,Ileibacterium,Akkermansia,and Lachnospiraceaand;decreased the abundance of Erysipelotrichaceae、Lactococcus 、 Dubosiella and Lactobacillus,and increased the proportion of Bifidobacteriaceae.Probiotics significantly increased the abundance of intestinal communities and the proportions of Bifidobacterium,Faecalibaculum and Ileibacterium in T2 DM mice;prebiotics increased the proportion of Bifidobacterium in the intestinal tract of T2 DM mice.Spearman’s correlation analysis showed that Streptococcus and Erysipelotrichaceae were positively correlated with corneal fluorescein sodium staining score and negatively correlated with tear secretion;Akkermansia was positively correlated with tear secretion and negatively correlated with serum IL-1β content and corneal fluorescein sodium staining score;Bifidobacterium,Ileibacterium and Lachnospiraceae were negatively correlated with serum IL-1β.(5)Purine metabolism,pyrimidine metabolism,and β-alanine metabolism were altered in T2 DM mice compared with the control group.Compared with mice in the T2 DM group,glycerophospholipid metabolism and sphingolipid signaling pathways were altered in the probiotic group;glycerophospholipid metabolism,biosynthesis of histidine and purine-derived alkaloids,biosynthesis of phenylpropanoids,isoflavone biosynthesis,and linoleic acid metabolic pathways were altered in the probiotic group.Spearman correlation analysis of differential metabolites and bacterial genera in control group mice and T2 DM mice showed that taurocholic acid was positively correlated with Streptococcus and negatively correlated with Akkermansia and Lactobacillus,etc.Deoxycholic acid was positively correlated with Erysipelotrichaceae but negatively correlated with Bacteroides.N1,N8 diacetyl spermidine was positively correlated with Muribaculaceae,Bacteroides and Akkermansia but negatively correlated with Lactococcus and Faecalibaculum.Conclusions:A T2 DM mouse model with dry-eye-like symptoms and associated pathological changes can be successfully established through a high-fat diet and STZ intraperitoneal injection.Both probiotics and prebiotics can improve dry eye symptoms and related pathological changes in T2 DM mice.The possible mechanisms include inhibiting cell apoptosis and immune cell infiltration in the intestinal tract and ocular surface of mice,enhancing antioxidant,anti-inflammatory and barrier function,and inhibiting TLR4/MyD88 inflammatory pathway.In conclusion,probiotics and prebiotics may improve dry eye symptoms and related pathological changes in T2 DM mice through the gut-eye axis.