Protective Effect Of Metformin On The Lacrimal Gland Of Diabetic Mice Induced By Streptozotocin

Background and objectiveDiabetes is a type of metabolic disease characterized by chronic increase in blood glucose level.With the increasing number of people suffering from diabetes worldwide,diabetes has increasingly become a serious disease threatening human health.Elevated blood sugar can cause changes in the systemic microenvironment,which in turn leads to many complications.Diabetic eye diseases mainly include diabetic cataract,diabetic retinopathy,neovascular glaucoma and diabetic dry eye.Recently,the effect of diabetes on the ocular surface or the anterior segment of the eye has become the focus of research.The main clinical manifestations and ocular pathological changes of diabetic ocular surface disease are dry eye,corneal neuropathy and corneal epithelial abnormality.Although the mechanism leading to these pathological changes in the anterior segment of eye has not been fully understood,studies have shown that these abnormalities are closely related to the dysfunction of the lacrimal glands in secreting tear fluid.Lacrimal glands are almond-shaped exocrine glands that mainly secrete the aqueous layer of the tear film on the ocular surface.The tear film plays a vital role in stabilizing the ocular surface.It provides comfort,mechanical,environmental,and immune protection for the ocular surface and forms part of the refractive system.The neuropeptides and protease inhibitors contained in the tear water secreted by the lacrimal glands are also very important for maintaining the dynamic balance of the ocular surface and promoting wound healing.The tear secretion of diabetic patients is significantly lower than that of normal people,while the tear film osmotic pressure is significantly increased.In addition,the stability of the tear film of diabetic patients is significantly reduced.Almost 50% of diabetic patients suffer from chronic dry eye disease.Diabetes can cause low-grade inflammation throughout the body and affect the function of the lacrimal glands.Compared with patients without diabetes,patients with type 1 and type 2 diabetes usually show more severe symptoms of dry eye.However,the mechanism by which diabetes affects the function of the lacrimal glands has not been fully elucidated.Therefore,there is an urgent need to determine the molecular mechanism that affects the function of the lacrimal gland.Metformin(Metformin)is a type of biguanide oral hypoglycemic agent for type 2diabetes that is ineffective in the treatment of simple diet and physical exercise.It has small side effects and good effects.But at present,there is no relevant research on the effect of metformin on lacrimal gland inflammation.For people taking metformin to treat diabetes,studying the effect of metformin on diabetic lacrimal glands and its mechanism may provide new ideas for the treatment of diabetic dry eye.This study aims to explore the changes of lacrimal gland function in mice and the related effects of metformin when diabetes occurs through transcriptome sequencing analysis,related animal experiments and molecular biological experiments,and to preliminary explore the role of PAMPs in it.Methods1.SPF-grade 2M-old male C57BL/6 mice were selected for streptozotocin intraperitoneal injection and high-fat diet to induce diabetic mouse models.Blood glucose,food intake,water intake and urine output were recorded during the period to observe the modeling situation.The effects of metformin on the intestines and lacrimal glands of diabetic mice were observed with the aid of intestinal permeability experiment and lacrimal gland tissue sections.2.The lacrimal gland samples of each group were collected for transcriptome sequencing.The sequencing results were analyzed by Dr.Tom platform and String database for bioinformatics analysis to screen the differentially expressed genes(DEGs),perform GO(gene ontology)functional enrichment analysis and KEGG pathway enrichment analysis,construct protein-protein interaction network analysis(PPI network analysis).Gene set enrichment analysis(GSEA)was performed on genes between groups.According to the analysis results,the potential protective mechanism and pathways of metformin on the lacrimal gland of diabetic mice were found.3.The accuracy of the sequencing results was verified by RT-PCR.SPSS 26.0statistical analysis software was used for statistical analysis.The t test was used for the test of gene expression levels between groups,and the difference was statistically significant with P <0.05.Results1.The fasting blood glucose level of the mice in the diabetes group was higher than 16.7mmol/L.Compared with the control group,the bodyweight of the mice in the diabetes group decreased significantly,and their food intake,water intake and urine output increased significantly.The symptoms of "three more and one less" were obvious.The model was successful.Intestinal permeability test results suggested that metformin could improve intestinal leakage in diabetic mice.Pathological sections of lacrimal glands of diabetic mice showed infiltration of lymphocytes and neutrophils around multiple lobular ducts,and even acinar atrophy,collapsed glandular duct structure,fibrosis between the lobules and within the lobules.Metformin could improve the above-mentioned pathological process.2.Compared with normal mice,diabetic mice had 683 DEGs.DEGs had concentrated functions.In terms of cell components,cell surface,extracellular space,cytoplasm and extracellular regions were significantly enriched.Molecular functions involved protein binding,protein dimerization activity,pheromone activity,and catalytic activity.As for biological processes,immune system,natural immune response,defense response to viruses,and cell response to interferon were mainly enriched.KEGG Pathway was mainly enriched in phagocytosis,antigen processing and presentation,cell adhesion,NOD-like receptor pathway,TNF signaling pathway,Th17 cell differentiation and lipopolysaccharide signaling pathway.3.Compared with the previous diabetic mice treated with metformin,there were163 DEGs.The vacuole membrane containing the symbiont was the main enriched cell component.GTPase activity and β-2-microglobulin binding were the main enriched molecular functions.Defense and immune responses were the main enrichment biological processes.NOD-like receptor pathway,TNF signaling pathway,saliva secretion,antigen processing and presentation,Th17 cell differentiation and lipopolysaccharide signaling pathway were the main enriched KEGG Pathway.4.The PPI protein interaction analysis obtained the hub genes between groups.The expression of these hub genes in diabetic mice showed an upward-regulated trend compared with the control group.The expression of hub genes between groups after receiving metformin treatment showed a downward-regulated trend compared with before.The results of PPI protein interaction analysis suggested that the hub genes in lipopolysaccharide signaling pathway were closely related to the Th17 signaling pathway.5.The RT-PCR results indicated that DEGs verified were consistent with the expression trends of the sequencing results,which proved that the sequencing results were true,accurate and highly reliable.Conclusion1.Diabetes can cause the intestinal permeability of mice to increase.Toxic substances such as LPS enter the lacrimal gland to cause inflammation and immune response,and the structure of the lacrimal gland is destroyed.Metformin can improve diabetic intestinal leakage,reduce the inflammation of the lacrimal gland,and maintain the normal structure of the lacrimal gland.2.LPS and metformin act on Toll-like receptor signaling pathway,Th17 signaling pathway,tumor necrosis factor superfamily and beta interferon,and jointly participate in the process of lacrimal gland inflammation and immune response in diabetic mice.