| Background and ObjectiveDiabetes is a worldwide epidemic,but is increasing rapidly in China.With the in-depth study of diabetes and related complications,more and more studies have begun to pay attention to diabetes combined with ocular surface lesions.Ocular surface lesions include changes in cornea,conjunctiva,tear film,sclera and microenvironment.According to the classification methods of organisms: kingdom,phylum,class,order,family,genus,species,and by comparing the obtained representative sequences with 16 S rRNA sequencing technology,bacteria classification information at the level of genus can be obtained.his study based on type 2 diabetes mellitus(Type2 diabetes mellitus,T2DM)people with normal glucose tolerance related parameters of ocular surface check,the number of bacteria in intestinal flora and ocular surface analysis,understand the in patients with type 2diabetes at present situation changes,and flora ocular surface disease diabetes cross-eyed table organization,the influence of intestinal flora and ocular surface flora.MethodsAccording to the inclusion and exclusion criteria,65 patients(130 eyes)with type 2 diabetes treated with insulin alone and 65 patients(130 eyes)with normal glucose tolerance were selected from the department of endocrinology,henan people's hospital,from march to June 2019.Patients with type 2 diabetes and those with normal glucose tolerance were divided into type 2 diabetes group(Db)and control group(Ct).Among them,96 cases of T2 DM group(Db)according to the results of fundus fluorescence angiography(FFA)divided into merger of T2 DM retinopathy(Db1)40 cases 80 eyes and T2 DM group of retinopathy(Db2),25 cases 50 eyes.Test the observation indexes respectively.The age of the type 2 diabetes group ranged from 41 to 52 years old,with an average age of 47.2±11.3 years.The gender composition was 48/17(male/female).The disease course ranged from 4 to 7 years,with an average course of 5.96±1.15 years.The control group ranged in age from 43 to 46 years,with an average age of 49.6±10.1 years and a gender composition of44/21(male/female).Part ?: Peripheral venous blood samples were collected for examination of related indicators.Blood routine,High sensitivity C reactive protein(hs CRP),Total bile acids(TBA),Glycosylated hemoglobin(HbA1c),IL-6,TNF-?,and Oral Glucose Tolerance Test were compared between the two groups.Changes of corneal nerve fibers under OGTT,ocular surface disease index(OSDI),tear break up time(BUT),Scherirmer I test(s It),and fluorescent staining of cornea(FL).Among them,OSDI score values can judge the severity of ocular surface symptoms.BUT reflects the change of tear film maintenance time,BUT < 10 s indicates that the tear film is unstable,and the higher the proportion of cases with BUT < 10 s,the worse the tear film function is.SIt reflects the change of tear secretion,the s It value of10-15mm/5min is normal,the s It < 10mm/5min indicates a decrease in tear secretion,and the higher the proportion of s It < 10mm/5min,the worse the tear secretion function is.FL score reflects the degree of corneal integrity damage,the higher the score,the more serious the damage.Images of corneal nerves were collected by confocal microscopy.Combined with software analysis,the length(mm)and density(number of strips)of corneal nerve fibers per unit area(mm2)and the density(number of strips)of corneal nerve branches per unit area(mm2)were observed to judge the corneal nerves.Part ?: fecal samples were collected and fecal DNA was extracted.16 S rRNA sequencing technology and multivariate statistical analysis tools were used to directly obtain the contents of different bacterial genera in the samples.The differences in the composition of intestinal flora between the two groups were compared at the genus level.The contents of genus and the proportion of each genus in the total bacteria were expressed as x±s.Part ?: conjunctival sac secretions were collected and the secreted DNA was extracted.16 S rRNA sequencing technology and multivariate statistical analysis tools were used to analyze the structural differences between the two groups at the phylum level and the genus level and the species related to the differences.Results Part ?: study on the present situation of ocular surface lesions in T2 DM patients1.Comparison of test indicators between the two groups showed that TBA in T2 DM group was significantly lower than that in the control group,and the difference was statistically significant(P<0.05).IL-6 in T2 DM group was significantly higher than that in control group,with statistically significant difference(P<0.05).There was no significant difference in PLR,hs CRP,TNF-levels between the two groups(P>0.05).2.Comparison of OSDI scores between the two groups showed that the score of T2 DM group was significantly higher than that of the control group,and the difference was statistically significant(P<0.001),the number and severity of ocular discomfort in the T2 DM group were higher.Compared with the control group,the BUT(9.69±2.38)and SIt(7.62±3.36)in the T2 DM group decreased significantly(P<0.001),and the tear film stability and tear secretion in the T2 DM group also decreased.Compared with the two groups,the F(5.47±2.65)L score of the T2 DM group was significantly higher,and the difference was statistically significant(P<0.05).The corneal damage degree of the T2 DM group was more serious than that of the control group.3.The proportion of BUT < 10 s in T2 DM group was 80.2%(52 eyes),the difference was statistically significant(P<0.05).The proportion of SIt results < 10 mm in T2 DM group reached 85.7%(56 eyes),with statistically significant difference(P<0.05).The eye surface function of T2 DM group was poor and the tear film was unstable.4.Comparison of corneal nerve examination results among the three groups:compared with the control group,corneal nerve branch density and corneal nerve fiber curvature in the Db2 group increased significantly(P< 0.05),and there was no statistically significant difference between corneal nerve fiber density and corneal nerve length(P> 0.05).T2 DM without diabetic retinopathy(DR2)did not present all corneal nerve damage.The corneal nerve length,corneal nerve fiber density and corneal nerve branch density in the DR1 group were smaller than those in the control group and the DR1 group(P< 0.05),while the corneal nerve fiber curvature was higher than that in the control group and the DR2 group(P< 0.05).The severe degree of corneal nerve injury in the T2 DM combined with DR group.Part ?: study on intestinal flora in patients with type 2 diabetes1.The total amount of intestinal bacteria in the two groups was the same,with no statistically significant difference(P> 0.05).Compared with the control group,the number of clostridium delicata and enterococcus in the T2 DM group was significantly increased(P< 0.05),while the number of bacteroides and bifidobacteria in the T2 DM group was significantly lower than that in the control group(P< 0.05).Compared with the control group,the contents of the genera prevotella and escherichia coli increased in the T2 DM group,while the contents of the spheroidal clostridium and lactobacillus decreased,but the differences were not statistically significant(P>0.05).2.In terms of the proportion of total bacteria in the two groups,compared with the T2 DM group,the proportion of bacteroides/clostridium spheroides(0.89±0.05)and bifidobacteria(0.54±0.13)in total bacteria was significantly decreased,while the proportion of clostridium tenaci(0.70±0.07)and enterococcus(0.59±0.15)in total bacteria was increased,with statistically significant differences(P < 0.05).3.The number of bacteroides and the proportion of bacteroides/globular clostridium in the total bacteria were significantly negatively correlated with HbA1C(P<0.05),while the number of clostridium delicata and enterococcus were significantly positively correlated with HbA1C(P < 0.05),while the number of other bacteria were not significantly correlated with HbA1 C.Part ?: The correlation study of ocular microflora in T2 DM patients1.Two groups of comparisons: alpha diversity comprehensive ACE,Chao1 index,Simpson index,Shannon index,OTU number,Fisher,a total of six indicators,the T2 DM group compared with healthy controls,the results show that type 2 diabetes group of ACE,Chao1 index,OTU number,Fisher is higher than the healthy control group,the differences between the two groups have statistical significance(P < 0.05),while the Simpson index and Shannon index there was no statistically significant difference with the healthy controls(P > 0.05).The abundance and diversity of ocular microflora in the type 2 diabetes group were higher than that in the healthy control group.2.Comparison of diversity between the two groups: the ocular surface microflora of the two groups could be distinguished from each other,with significant differences.The microflora of the type 2 diabetes group was more dispersed,while the samples of the healthy control group were more concentrated.Compared with the two groups,the diversity of the type 2 diabetes group was significantly different(P<0.05),and the difference was more obvious on the horizontal axis of PC2 and NMDS2.3.Differences in the group structure of the ocular surface bacteria of the two groups at the phylum level: Bacteroidetes,Actinobacteria and Proteobacteria were mainly found at the phylum level of the healthy control group,while Firmicutes in the samples of type 2 diabetes group were higher,with greater differences between samples.Comparison of mean abundance of samples in the two groups: firmicutes and Thermus with the highest proportion in the type 2 diabetes group were significantly higher than the healthy control group,Fusobacteria was slightly higher than the healthy control group,and actinomycetes and proteobacteria were significantly lower than the healthy control group.Lef Se analysis showed that the relative abundance of firmicutes and Deinococcus and the genus anemophilus in the type 2 diabetes group was higher than that in the healthy control group,while the relative abundance of proteobacteria and bacteroidetes was lower,with statistically significant differences(all P<0.05).Comparison of species abundance between the two groups: the proportion of firmicutes in the type 2 diabetes group was significantly higher than that in the healthy control group(P=0.005),while the proportion of proteobacteria was significantly lower than that in the healthy control group(P=0.003),with statistically significant differences.4.Structural differences of ocular surface bacteria in the two groups at the genus level: comparison of mean abundance of samples in the two groups: Faecalibacterium,pyreomyces,bacteroidetes and Neisseria with the highest proportion in the type 2diabetes group were higher than those in the healthy control group,while actinomyces were lower than those in the healthy control group;The relative abundance of Paenibacillus and Ruminococcaceae in the type 2 diabetes group was higher than that in the healthy control group,while the relative abundance of Enhydrobacter and its class and phylum was lower than that in the healthy control group,with statistically significant differences(all P<0.05).Comparison of species abundance between the two groups: the proportion of bacillus and Paenibacillus in the type 2 diabetes group was significantly higher than that in the healthy control group(P=0.009;However,Enhydrobacter was significantly lower than that of the healthy control group(P=0.004),with statistically significant differences.ConclusionPatients with type 2 diabetes have increased ocular surface lesions,and are prone to tear film stability decline,tear secretion decrease,corneal epithelial injury and corneal nerve abnormality.These abnormalities may be caused by changes in the composition ratio of intestinal flora,while type 2 diabetes may cause changes in the composition of ocular surface flora. |
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