| Aim:To establish a diabetic rat xerostomia model and observe its salivary gland structure and function changes and differences of oral flora.Explore the effects of artesunate on xerostomia and oral flora changes induced by diabetes mellitus.Methods:Forty male SD rats of six-week-old were selected and 32 diabetic rats were established by intraperitoneal injection of streptozotoein.The diabetic rats were randomly divided into four groups,8 in each group,and 8 in the Normal group.After the intervention,the insulin treatment group(DM+INS)was administered by abdominal subcutaneous injection(6 U/kg per day),artesunate intervention group(DM+ART)by daily intragastric administration,DM+ART 50 mg/kg group by 50 mg/kg body weight,DM+ART 100 mg/kg group by 100 mg/kg body weight,DM group and normal Normal group by no intervention.The body weight and fasting blood sugar of rats were measured weekly during the experiment.At the end of the 4th week of intervention,rats in each group were measured for 12 hours of food intake,drinking water,urine and feces;stimulated salivary flow rate induced by pilocarpine(5 mg/kg)for 5 minutes;oral swabs of rats in each group were taken,total DNA was extracted,and 16S RNA V 3+V 4 region was amplified with 97%consistency clustering and species annotation based on Silva database to study the changes of oral flora of rats in each group.At the end of the experiment,all animals were sacrificed,abdominal aorta blood was taken for laboratory examination,and the histological changes of submandibular gland,buccal mucosa,tongue and pancreas were observed.The distribution and expression levels of aquaporin 5(AQP5)and inducible nitric oxide synthase(NOS2)in submandibular gland tissues were analyzed by immunohistochemistry and PCR.The ultrastructural changes of submandibular gland acinar cells were observed by transiission electron microscopy.Results:1.At the end of the experiment,the average body weight of the normal Normal group was significantly higher than that of all other groups(P<0.05);the DM+INS group and the DM+ART 50 mg/kg group were significantly higher than the DM group.2.The final blood glucose Normal group was 6.0±0.5 mnol/L significantly lower than all other groups.The DM group had the highest blood glucose level(39.5±7.2 mmol/L),the DM+INS group(26.1 ±3.8 mmol/L)and DM.The blood glucose level of the+ART 100 mg/kg group(29.9±3.6 mmol/L)was significantly decreased(P<0.05).3.The amount of water in diabetic rats(124.63±17.45 ml)increased significantly,and the amount of water consumed after artesunate intervention decreased(P<0.05).4.The stimulating salivary flow rate of DM group was 1.5±0.73 ?l/min,which was significantly lower than that of Normal group,41±25.57 ?/min,and increased significantly after 4 weeks of intervention(P<0.05).Among them,DM+ART 50 mg/kg group showed the best effect in salivay secreting(22.78±5.57 ?l/min);5.The oral flora of diabetic rats changed significantly,such as Proteus increased sharply to about twice that of normal rats(P<0.05).After oral administration,the oral flora of each group changed to varying degrees,of which DM+ART 50 mg The proportion of the pre-oral 5 dominant bacteria in the/kg group was the closest to that in the normal group.6.H&E staining results Submandibular gland:The submandibular gland acinar cells in the DM group were shrunk and clustered,and scattered vacuoles were observed in the cytoplasm,and the secretory granules were obviously condensed;DM+ART 50 mg/kg group and DM+INS group acinus The atrophy and accumulation of cells improved significantly,and the secretory granules were full.Buccal mucosa:in the DM group,the buccal mucosa became thinner,the acanthal cells decreased,the granular layer disappeared,and the basal cells lost polarity.After the artesunate intervention,the acanthocytes increased,and the basal cells partially disappeared;DM+In the INS group,the epithelial nails appeared deep into the lamina propria.Tongue:The atrophy of the gingival papilla of the DM group is "spike-like" and the top horny layer is thickened.The epithelial keratinized layer of the tongue and abdomen is thickened obviously,the nail process is jagged into the lamina propria,the granular layer cells are slightly swollen,and the acanthosis is hyperplasia;the DM+INS group and the DM+ART 100 mg/kg group are the lateral margin bacteria.The degree of nipple atrophy is improved and the top stratum corneum is thinned.The abnormal proliferation of lingual and abdominal mucosal epithelial nails in DM+ART 100 mg/kg group was improved.7.IHC and PCR results showed that AQP5 was highly expressed in the subrnandibular gland acinar cells of rats,and the positive expression was concentrated on the cell membrane;the expression of AQP5 in diabetic rats was significantly decreased(P<.0.05);the expression was increased after insulin intervention,but the expression site Dispersed in the cytosol,and the positive rate on the membrane is still low;the expression of AQP5 is the closest to normal after the low dose of artesunate.The results of RT-PCR also indicated that the relative expression of AQP5 mRNA was significantly increased in the diabetic rats after low-dose artesunate intervention(P<0.05),but still lower than the Normal group.There were no significant differences in relative expression of NOS2 mRNA between normal and diabetic non-intervention groups,but the expression of artesunate was significaritly increased after intervention(P<0.05).8.Ultrastructural observation:The karyocysts of the submandibular gland acinar cells in the DM group were heteromorphic,and a large number of lysosomes were observed in the cytoplasm.The endoplasmic reticulum was dilated,the mitochondrial morphology was swollen,and the mitochondrial raft structure was broken.Autophagic lysosomes were observed.The DM+ART 50 mg/kg group showed significant improvelent in nuclear irregularity,the number of mitochondria increased significantly,mitochondria were clearly visible,and the rough endoplasmic reticulum was developed and increased significantly;lysosomes were found in the cytoplasm of DM+ART 100 mg/kg group.Scattered distribution.The improvement in the DM+INS group was not obvious.Conclusions:1.The salivary secretion of diabetic rats is significantly decreased,and the structure of acinar cells in the submandibular gland is destroyed.2.Artesunate may have a certain hypoglycemic effect,and the hypoglycemic effect is related to the dose.The lower dose of artesunate has a stronger hypoglycemic effect.3.Artesunate intervention can improve the secretion of stimulating saliva in diabetic rats;the appropriate dose of artesunate may have protective effects on submandibular gland cells in diabetic rats.4.Artesunate intervention can significantly improve the expression and transport of AQP5 mRNA and protein in the submandibular gland of diabetic rats;artesunate application may not improve the oxidative stress in the submandibular gland of diabetic rats.5.The difference between oral and dominant bacteria in normal and diabetic rats was significant.Proteus was the most obvious in the oral flora of diabetic rats.The oral dominant bacteria were close to the normal group after low dose of artesunate. |
PDF Full Text Request