| OBJECTIVESTo establish diabetic rat model and observe the structural changes of the lacrimal gland and eyeball as well as analyze tear secretion.To evaluate the use of artesunate and its effect to explore the potential mechanism of action.METHODS AND MATERIALSSix week old male Sprague-Dawley rats were provided and after one-week of adaptive feeding,diabetes was induced by intraperitoneal injection of streptozotocin(STZ)60 mg/kg body weight.The healthy control groups were injected with sodium citrate buffer only.The fasting blood glucose levels were detected on 3 rd,7th and 14th day with a glucose meter test to verify diabetic status.Fasting hyperglycemia over more than 16.7 mmol/L was considered to confirm diabetic model.On the 15th day diabetic groups were further divided into four groups:Diabetic insulin(6 U/kg body weight,a daily subcutaneous injection)intervention group,diabetic artesunate 50 mg/kg intervention group,and diabetic artesunate 100 mg/kg intervention group(oral administration once per day),and diabetes mellitus group.Throughout our study,for consecutive four weeks,the fasting blood glucose and body weight were detected in every week from each diabetic rat indicating hyperglycemia and type 1 diabetes.At the end of the fourth week,the metabolism data of all rats were measured,including the data of food,water,urine,and feces.At the same time,the amount of tear secretion of the rats was measured with phenol cotton thread,and the symptoms of dry eyes were evaluated.All animals were anesthetized and sacrificed at the end of 4th week.Each of the tissues was divided for further laboratory investigation as follows:histopathological examination,ultra-structural examination,immunohistochemical analyses for NFκB1 and TNF-α protein expression and RT-PCR.Blood from abdominal aorta and urine from the bladder were collected immediately after they were sacrificed into sterile tubes for specialized laboratory examination.Statistics comparisons(SPSS version 22.0)were done using one-way analysis of variance(ANOVA)followed by Tukey’s test.RESULTS1.The tear secretion by phenol red thread test showed significantly reduced tear production on the diabetic group(1.362±0.94 mm)compared with the control group(3.937±1.522 mm)(P<0.05).After drug intervention,tear secretion on ART 100 mg/kg was significantly higher than insulin intervention and ART 50 mg/kg group(*P≤0.05).2.The average weight of lacrimal glands in normal rats was 484.227±51.86 mg,which was significantly higher than 238.085±37.008 mg in the diabetic group.Weight of lacrimal gland of insulin intervention was significantly higher than diabetic group whereas the weight of lacrimal gland of ART 50 mg/kg and ART 100 mg/kg intervention group was significantly lower compared to the diabetic group(*P≤0.05).The lacrimal gland weight/body weight of the control group(1.23±0.5 mg/g)was significantly higher than that of the diabetes group(1.07±0.4 mg/g).All intervention groups were significantly lower than diabetic group(*P<0.05).3.H&E staining lacrimal gland:Compared to the control group,diabetic group and intervention diabetic group showed an abnormal lobular pattern,acinar atrophy,fibrosis,inflammatory cells,and disorganized acinar and ductal cells were observed.Cornea:The total thickness of diabetic cornea was significantly higher,and adhesion deficiency in the corneal basal cells and Bowman’s membrane of STZ rats was observed as compared to in those of normal rats than that of the normal group.Insulin and ART 100 mg/kg intervention group have thinner corneal stroma compared to the diabetic group.Lens:The number of cell nuclei was remarkably decreased,and a large number of distinctive vacuolar changes in the cortical region was detected in the diabetic group.On ART 100 mg/kg and insulin intervention group,the number of the cell nuclei was remarkably increased,and fewer vacuoles were observed in the cortical region.Retina:The morphological changes of inner nuclear layer(INL),outer nuclear layer(ONL),and degenerated ganglionic cells and partial loss of outer plexiform layer(OPL)in the diabetic group.4.Immunohistochemistry and RT-PCR analysis:The expression levels of NFKB1and TNF-α in the lacrimal gland tissues of DM rats were significantly higher than those of normal rats.The expression levels of NFκB1and TNF-α in the lacrimal gland tissues of DM rats were significantly lower after the intervention of ART and INS.ART 100 mg/kg also significantly reduced the expression of NFκB1,but the down-regulation of TNF-α in the lacrimal glands of DM rats was not significant(*P>0.05).The results of RT-PCR showed that the mRNA levels of inflammatory factors NFKB1 and TNF-α in lacrimal gland tissues of DM rats were significantly higher than those of normal rats.The mRNA levels of NFκB1 and TNF-α decreased significantly after insulin and ART intervention(*P<0.05).5.Transmission electron microscope(TEM)revealed the fragmentation and shrinkage of nuclei,swelling of rough endoplasmic reticulum,secretory granules were smaller in size and disorganized mitochondria in the diabetic groups.Lysosomes and autolysosomes,lipid droplets in the cytoplasm were observed in the diabetic group.Nuclear pyknosis,chromatin distribution was basically uniform and some mitochondrial crista structures were destroyed,intracellular lysosomes were increased,numerous lipid droplets were observed in the cytoplasm of diabetic insulin intervention group.On diabetic ART 50 mg/kg intervention group,mitochondria and rough endoplasmic reticulum number increased obviously,mitochondrial cristae is clearly visible,the nucleus has no obvious abnormality,and nucleolus were uniformly distributed,abundant rough endoplasmic reticulum and observed lysosomes.On diabetic ART 100 mg/kg intervention group,fragmentation and shrinkage of nuclei,mitochondria were more developed,and lysosome was visible on the cytoplasm.CONCLUSIONThe structural changes of the exorbital lacrimal gland and eye ball in diabetic rats was seen.The use of Artesunate showed decrease in the local inflammatory expression of NFκB1 and TNF-α,thus improving structural changes and regulating the tear secretion of diabetic rats. |
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