CN115531376A - Novel application of canagliflozin in flora regulation - Google Patents

Abstract

The invention relates to a new application of a medicament, in particular to a new application of canagliflozin in flora regulation, which is shown in regulation of intestinal flora and ocular surface flora of type 2 diabetes patients. The canagliflozin treatment reduces FBG, GSP and body weight of a T2DM patient, and changes the composition of intestinal flora: at the phylum level, actinomycetemcomita after intervention is significantly lower than before intervention, and bacteroidetes is increased, at the genus level, solospirillum after intervention is significantly higher than before intervention, bifidobacterium, colinerium are decreased; canagliflozin reduces the occurrence of ocular diseases by reducing the opportunistic pathogenic flora of the ocular surface and increasing the protective flora, and the altered ocular surface flora reduces the inflammatory response by increasing amino acid biosynthesis.

Description

Novel application of canagliflozin in flora regulation

Technical Field

The invention relates to a new application of a medicament, in particular to a new application of canagliflozin in the regulation of intestinal flora and ocular surface flora of patients with type 2 diabetes.

Disclosure of Invention

With the development of society, especially the progress of science and technology, the rapid development of social productivity is greatly promoted, so that the life of people is greatly improved; social progress provides good basic environment for people's physical and mental lives, and along with people enjoying increasingly abundant physical lives, many diseases come with the people, and the people gradually show a wide range and a low age trend, and typical representatives of the diseases are diabetes, hypertension and the like. Diabetes Mellitus (DM) is becoming a serious chronic disease affecting the health of the nation. It is estimated that the prevalence of diabetes in the 20-79 year old population worldwide is 10.5% (about 5.366 million people) in 2021 and will rise to 12.2% (about 7.832 million people) by 2045. The number of Chinese diabetic patients is the most, more than 1 hundred million and 4000 million people in 2021, and more than 1 hundred million and 7400 million people in 2045 ^] 。

Diabetes is a metabolic disease characterized by hyperglycemia. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. The long-standing hyperglycemia causes chronic damage and dysfunction of various tissues, particularly eyes, kidneys, heart, blood vessels and nerves. Diabetes is further classified as type 1 or type 2 diabetes. There is significant genetic heterogeneity in both type 1 and type 2 diabetes. Diabetes has family incidence tendency, and 1/4-1/2 patients have family history of diabetes. Clinically, at least 60 genetic syndromes may be accompanied by diabetes. Type 1 diabetes has multiple DNA sites involved in onset, with the DQ site polymorphism in HLA antigen genes being the most closely related. In type 2 diabetes, a variety of well-defined genetic mutations have been found, such as the insulin gene, insulin receptor gene, glucokinase gene, mitochondrial gene, and the like. Type 2 diabetes, also known as non-insulin dependent diabetes mellitus, is mainly characterized in that a human body can produce insulin, and cells can not make corresponding response to the insulin, so that the insulin can not play a corresponding role in the body. A class of diseases commonly caused by insulin resistance, combined with relative insulin hyposecretion. The etiology and pathogenesis of type 2 diabetes are relatively complex, and are not completely clarified so far, but the deficiency or serious insufficiency of insulin in a body is probably caused by the defects of insulin resistance and insulin secretion of peripheral tissues caused by genetic factors and environmental factors, so that the utilization rate of glucose is reduced, hyperglycemia is caused, and then diabetes is developed.

Diabetic retinopathy becomes one of the most common and most severe microvascular complications of diabetes. Domestic data show that the prevalence of diabetic retinopathy, non-proliferative diabetic retinopathy and proliferative diabetic retinopathy is 18.45%, 15.06% and 0.99%, respectively.

Canagliflozin, under the trade name Inovokana, is the first SGLT2 inhibitor approved by the FDA for the treatment of type 2 diabetes in adult patients. The sodium-glucose co-transporter is a glucose transporter, has two subtypes, namely SGLT1 and SGLT2, which are respectively distributed in small intestinal mucosa and renal tubules and can transport glucose into blood. Canagliflozin can inhibit SLCT2, so that glucose in renal tubules cannot be smoothly reabsorbed into blood and is discharged with urine, thereby reducing blood glucose concentration.

Therefore, the new application of the exploogliflozin in the aspect of treating the complications caused by diabetes is a problem worthy of research.

Disclosure of Invention

The invention aims to provide a new application of canagliflozin in treating intestinal flora and ocular surface flora regulation of type 2 diabetes patients.

The purpose of the invention is realized as follows:

the canagliflozin can be used for treating the blood sugar of a type 2 diabetes patient and simultaneously has new application of regulating intestinal flora;

the canagliflozin treatment reduces FBG, GSP and body weight of a T2DM patient and changes the composition of intestinal flora; at the phylum level, actinomycetomes were significantly lower after intervention than before intervention, while bacteroidetes were increased, at the genus level, drospirillum was significantly higher after intervention than before intervention, and bifidobacteria, colinlia were decreased.

The canagliflozin can be used for treating the blood sugar of a type 2 diabetes patient and has new application of adjusting ocular surface flora;

canagliflozin reduces the incidence of ocular diseases by reducing the opportunistic pathogenic flora of the ocular surface and increasing the protective flora, and the altered ocular surface flora reduces the inflammatory response by increasing amino acid biosynthesis.

Has the positive and beneficial effects that: according to the invention, through a large number of clinical case analysis summaries, the research summary analysis of the gliflozin applied to clinically treating the blood sugar reduction of the type 2 diabetes patients well proves the new application of the gliflozin, which is mainly reflected in the new application of the gliflozin in the aspect of regulating intestinal flora and ocular surface flora; provides good basic research for treating the diabetic complication by adjusting the flora in the two aspects.

Drawings

FIG. 1 is a graph I comparing the diversity of intestinal flora alpha before and after treatment in example 1 of the present invention;

FIG. 2 is a graph II comparing the diversity of intestinal flora alpha before and after treatment in example 1 of the present invention;

FIG. 3 is a graph showing a comparison of the intestinal flora beta diversity before and after treatment in example 1 of the present invention;

FIG. 4 is a graph II comparing the intestinal flora beta diversity before and after treatment in example 1 of the present invention;

FIG. 5 is a first analysis of the differences in intestinal flora structure before and after treatment according to example 1 of the present invention;

FIG. 6 is a second analysis of the difference in intestinal flora structure between before and after treatment according to example 1 of the present invention;

FIG. 7 is a third analysis of the difference in intestinal flora structure before and after treatment in example 1 of the present invention;

FIG. 8 is a fourth analysis of the difference in intestinal flora structure before and after treatment according to example 1 of the present invention;

FIG. 9 is a fifth analysis of the difference in intestinal flora structure between before and after treatment according to example 1 of the present invention;

FIG. 10 is a sixth analysis of the difference in intestinal flora structure before and after treatment according to example 1 of the present invention;

FIG. 11 is a diagram showing the analysis of the function prediction of the bacterial flora before and after the treatment in example 1 of the present invention;

FIG. 12 is a graph showing a comparison of the ocular surface flora alpha diversity of type 2 diabetic patients before and after intervention according to example 2 of the present invention;

FIG. 13 is a graph II comparing the diversity of the ocular surface flora alpha of type 2 diabetic patients before and after intervention according to example 2 of the present invention;

FIG. 14 is a graph comparing the ocular surface flora beta diversity of type 2 diabetic patients before and after intervention according to example 2 of the present invention;

FIG. 15 is a first analysis chart of the ocular surface flora composition of type 2 diabetic patients before and after intervention in example 2 of the present invention;

FIG. 16 is a graph of analysis of ocular surface flora composition of type 2 diabetic patients before and after intervention in example 2 of the present invention;

FIG. 17 is a first analysis chart of the ocular surface flora Mann-Whitney U test of type 2 diabetic patients before and after intervention in example 2 of the present invention;

FIG. 18 is a chart of the second Ocular surface flora Mann-Whitney U test analysis of type 2 diabetic patients before and after intervention in example 2 of the present invention;

FIG. 19 is a graph of LEfSe analysis of ocular surface flora of type 2 diabetic patients before and after intervention in example 2 of the present invention;

FIG. 20 is a graph of analysis of the ocular surface bacterial group random-forest of type 2 diabetic patients before and after intervention in example 2 of the present invention;

FIG. 21 is a diagram of the analysis of the function prediction of ocular surface flora of type 2 diabetic patients before and after intervention in example 2 of the present invention.

Detailed Description

Example 1

The canagliflozin can be used for treating the blood sugar reduction of a type 2 diabetes patient and has a new application of regulating intestinal flora;

1. object selection

1. 25T 2DM patients were selected from the clinic of the Endocrinology department in the laboratory from 11 months to 2021 months in 2020. 3 of the medicines were not used, 1 of other oral hypoglycemic medicines was used, 2 of the diarrhea patients were used, and 19 of the patients who could be analyzed for intestinal flora in this study were used.

2. T2DM diagnostic criteria: according to the 2011 diagnosis standard of the world health organization, T2DM can be diagnosed by one meeting the following conditions (1) typical symptoms of diabetes (polydipsia, polyphagia, polyuria and weight loss) with blood sugar of more than or equal to 11.1mmol/L or fasting blood sugar of more than or equal to 7.0mmol/L at any time; (2) The blood sugar of the Oral Glucose Tolerance Test (OGTT) is more than or equal to 11.1mmol/L after 2 hours.

3. And (3) inclusion standard: (1) age 30 to 70 years; (2) The diabetes diagnosis standard of the world health organization in 2011 is met; (3) Newly developed diabetes or T2DM patients without any oral hypoglycemic agent.

4. Exclusion criteria: (1) Type 1 diabetes patients, gestational diabetes, other special type diabetes; (2) oral hypoglycemic agents are used; (3) Patients who had taken antibiotics, traditional Chinese medicines, probiotics or any other drug that may affect the intestinal flora for the first 3 months; (4) Suffering from severe organic diseases such as cancer, hepatitis, cirrhosis, cerebral infarction, etc.; (5) Patients with persistent vomiting or suspected digestive tract obstruction, and digestive system surgical operations such as gastrectomy, fundoplication, colostomy, etc.; (6) Can be used for treating other endocrine diseases such as hyperthyroidism, hypothyroidism, polycystic ovary syndrome, etc.; (7) alcoholism; (8) People who cannot take care of themselves, cannot clearly recall and answer questions or have obvious dyskinesia due to physical disability or other reasons; (9) sufficient time cannot be guaranteed to participate in the project.

5. Patients who meet the above standards all adopt a voluntary principle, and inform the patients of the purpose, process, result, attention and related risks before collecting specimens, sign an informed consent, and quit the research or refuse to participate at any time. The study was approved by the ethical committee of the experimental unit. All subjects obtained informed consent and signed an informed consent.

2. The experimental method comprises the following steps:

1. data collection: the patient fills in a basic information questionnaire including name, gender, age, and eating habits. Fasting plasma glucose (FBG), glycated Serum Protein (GSP), total Cholesterol (TC), triglycerides (TG), high Density Lipoprotein (HDL), low Density Lipoprotein (LDL), and early morning Fasting subject were measured before and after 4 weeks of canagliflozin treatment to determine subject Body weight, body Mass Index (BMI), waist-hip ratio (Waist-hip ratio), systolic Blood Pressure (SBP), diastolic Blood Pressure (DBP).

2. Experiment design: the patient takes canagliflozin 100mg once a day in a single dose. Comparing the effect of 4 weeks on blood glucose and body weight and changes in intestinal flora composition in type 2 diabetic patients.

3. Collecting a fecal specimen: stool samples volunteers were asked to collect fresh stool in the morning using a sterile spoon into 8ml sterile cryovials and immediately placed in a-80 ℃ freezer for storage. The patient should wash his hands prior to sampling and perform other necessary cleaning measures, and the collection container delivered to the patient identifies the patient's name and date. Avoid the faecal sample to be contaminated by urine during collection.

4. Bioinformatics analysis and statistics: (1) PCR was performed using the V3-V4 region of the 16SrRNA gene as the target amplification region. Preparing a sequencing library, and performing high-throughput sequencing analysis; (2) Screening the obtained sequences, and then merging and dividing OTU; (3) Evaluating the diversity level of each sample according to the abundance distribution of the OTU in different samples; (4) Analyzing the specific composition of each group of samples at different classification levels; (5) Further measuring the flora structure difference among different groups and species related to the difference by using a multivariate statistical analysis tool; (6) Data processing and statistical analysis used Excel 2021 software and SPSS 25.0 software. Normal distribution measurement data is expressed by Mean + -SD, group comparison is expressed by paired t test, non-normal distribution measurement data is expressed by median (interquartile distance), and group comparison is expressed by rank sum test. P <0.05 is statistically significant.

3. And (3) analyzing an experimental result:

1. comparison before and after administration

Before and after administration, the data such as waist-hip ratio, SBP, DBP, TC, TG, HDL, LDL and the like have no statistical significance (P is more than 0.05). The data of the weight, BMI, FBG and GSP have statistical significance (P is less than 0.05), and are shown in the following comparison table of indexes before and after treatment:

2. analysis of intestinal flora changes before and after drug administration

(1) α diversity variation: the curve of the accumulation of species, the abscissa is the sample size and the ordinate is the number of OTUs, i.e. the number of species, gradually becomes flat as the sample size increases, which shows that the increase of new species has no obvious change when the sample size is increased again, as shown in fig. 1. The ACE, chao 1, shannon and Simpson diversity indices of the faecal flora of type 2 diabetic patients before intervention (pre-I) and after intervention (post-I) were not significantly different in both faecal samples, indicating that there was no significant change in the alpha diversity of the faecal flora after intervention compared to before intervention (P > 0.05), as shown in figure 2, pre-I: before intervention; post-I Dry prognosis.

(2) β diversity change: based on PCoA analysis of Weighted-UniFrac distance, finding out the tendency that the stool samples before and after intervention gather to different positions in the graph, as shown in figure 3; adonis analysis based on Weighted-UniFrac distance also found that there was some overall difference in fecal flora before and after intervention (P = 0.059), as shown in fig. 4. These results indicate that the fecal flora has changed to some extent on the whole after intervention, pre-I before intervention; post-I Dry prognosis.

Species composition difference analysis of intestinal flora

Before and after intervention, the fecal flora takes Firmicutes, bacteroidetes and Proteobacteria as dominant phyla, as shown in fig. 5; on the genus level, the faecal flora before and after intervention with the genera Bacteroides (Bacteroides), faecalibacterium and subdoligranum as dominant genera is shown in fig. 6.

And (4) selecting the dominant bacterial groups with obvious difference before and after intervention by adopting Mann-Whitney U test. At the phylum level, bacteroidetes phyla was found to be significantly higher after intervention than before intervention, and actinomycetemcomia (Actinobacteriota) was found to be significantly lower after intervention than before intervention, as shown in fig. 7; at the genus level, bifidobacteria (bifidobacteria), ruminococcus _ torques _ group and collina (collinesla) were significantly lower after intervention than before intervention and Lachnospira (Lachnospira) were significantly higher after intervention than before intervention, as shown in fig. 8.

Based on LEfSe analysis, bacterial genera were selected that showed significant differences in fecal flora before and after intervention. The genera bifidobacterium, colepsy, gemella, saccharomyces and granuligera were found to be significantly higher before intervention than after intervention, as shown in figure 9.

Using random-forest analysis, 22 OTUs were found to differ in fecal flora before and after intervention, with 9 OTUs being higher after intervention than before intervention, and belonging to respectively phocae, escherichia (colibacillus), oscillatoria (Oscillibacter), oscillospora _ UCG-003, ruminococcus _ UBA1819, alistipes, and an unclassified genus of the bacteroidetes and the anaspirillaceae families; the remaining 13 OTUs, which were superior to the dry prognosis before the intervention, belong to the genera Streptococcus digestions (Peptostreptococcus), oribacterium, saccharomyces onanals, gemelal, granulicatella, eggerthella, streptococcus (Streptococcus), coriolus, coprococcus, blauia, eubacterium _ villi _ group and Ruminococcus _ torques _ group, respectively, as shown in FIG. 10, pre-I: before the intervention; post-I Dry prognosis.

Functional predictive analysis

Fecal flora 16SrRNA sequencing data is subjected to PICRUSt function prediction based on a KEGG pathway database, and then through LEfSe analysis, metabolic pathways (L3 level) with significant difference in fecal flora before and after intervention are selected, wherein the selected differential metabolic pathways meet the condition that the P value is significant and LDA is more than or equal to 2.0 (figure 4). Compared with the pre-intervention pathway, the pathways such as Calcium signaling pathway (Calcium signaling pathway), apoptosis (Apoptosis), protein degradation and absorption (Protein degradation and absorption), N-polysaccharide biosynthesis (N-glycobiosynthesis) and the like of a T2DM patient are obviously increased after the intervention, the pathways such as Nitrotoluene degradation (Nitro toluene degradation), glycolytic carbohydrate neogenesis (glycogenogenesis) and Proteasome (Proteasome) are obviously reduced, and pre-I is that before the intervention; post-I Dry prognosis.

Summary of the invention

The canagliflozin treatment reduces FBG, GSP and body weight of a T2DM patient and changes the composition of intestinal flora. At the phylum level, actinomycetomes were significantly lower after intervention than before intervention, while bacteroidetes were increased, at the genus level, drospirillum was significantly higher after intervention than before intervention, and bifidobacteria, colinlia were decreased.

The experiment proves that the composition of intestinal flora species is changed while the treatment of the Mingkagliflozin reduces the blood sugar and the weight of a T2DM patient; the altered gut flora reduces inflammatory responses and insulin resistance by increasing SCFAs and/or calcium signaling pathways in the gut, thereby improving blood glucose and reducing weight.

Example 2

The gliflozin can be used for treating the blood sugar reduction of a type 2 diabetes patient and has a new application of regulating ocular surface flora;

1. object selection

21T 2DM patients were selected for outpatient clinics of the endocrinology department in the laboratory from 11 months to 5 months in 2020 to 2021. Subjects must meet all criteria for inclusion. Inclusion criteria were as follows: (1) age 30 to 70 years; (2) According to the 2011 diagnosis standard of the world health organization, a T2DM patient without any oral hypoglycemic agent can be diagnosed when one of the following conditions is met: (1) has typical symptoms of diabetes (polydipsia, polyphagia, polyuria and weight loss), and the blood sugar at any time is more than or equal to 11.1mmol/L or the fasting blood sugar is more than or equal to 7.0mmol/L; (2) an Oral Glucose Tolerance Test (OGTT), wherein the blood sugar is more than or equal to 11.1mmol/L after 2 hours; (3) there is no history of serious systemic and ocular diseases. Exclusion criteria: (1) use of antibiotics or glucocorticoids for approximately 6 months; (2) using the eye drops in about 6 months; (3) wearing contact lenses or cosmetic pupils; (4) the prior eye related operation history; (5) Suffering from severe organic diseases such as cancer, hepatitis, cirrhosis, cerebral infarction, etc.; (7) the subject is pregnant or lactating; (8) Other endocrine diseases such as hyperthyroidism, hypothyroidism and polycystic ovary syndrome; (9) alcoholism; (10) Physically disabled, or otherwise disabled people who cannot take care of their lives, cannot recall and answer questions clearly, or have significant movement impairment. The study was approved by the ethical committee of the experimental unit. All subjects obtained informed consent and signed an informed consent.

2. Experimental methods

1. Data collection: the patient fills out a basic information questionnaire including name, sex, age, history of systemic or ocular diseases and medication history. Fasting plasma glucose (FBG), glycated Serum Protein (GSP), total Cholesterol (TC), triglycerides (TG), high Density Lipoprotein (HDL), low Density Lipoprotein (LDL), and early morning Fasting subject were measured before and after 4 weeks of canagliflozin treatment to determine subject Body weight, body Mass Index (BMI), waist-hip ratio (Waist-hip ratio), systolic Blood Pressure (SBP), diastolic Blood Pressure (DBP).

2. Experiment design: the patient takes canagliflozin 100mg once a day with a single medicine. The effect of 4 weeks of treatment on blood glucose and changes in ocular surface flora in patients with T2DM were compared.

3. Collecting an ocular surface flora sample: when the conjunctival sac specimen of the testee is collected, the eyelid skin is firstly cleaned, the 0.5 percent proparacaine hydrochloride eye drops are used for local anesthesia of the ocular surface, the disposable sterile dry cotton swab is used for collecting the specimen at the conjunctival sac of the testee, and the eyelashes and the outer eyelid skin are forbidden to be contacted, so that the pollution is prevented. The collected cotton swabs are independently placed in a 1.5ml Eppendorf sterilization tube and are placed in a refrigerator at-80 ℃ within 5-10 minutes until DNA is extracted.

4. Bioinformatics analysis and statistics: (1) PCR was performed using the V3-V4 region of the 16SrRNA gene as the target amplification region. Preparing a sequencing library, and performing high-throughput sequencing analysis; (2) Screening the obtained sequences, and then merging and dividing OTU; (3) Evaluating the diversity level of each sample according to the abundance distribution of the OTU in different samples; (4) To pairAnalyzing the specific composition of each group of samples at different classification levels; (5) Further measuring the flora structure difference and species related to the difference among different groups by a multivariate statistical analysis tool; (6) Data processing and statistical analysis used Excel 2021 software and SPSS 25.0 software. Normal distribution measurement data is expressed by Mean plus or minus SD, the comparison among groups is expressed by a pairing t test, the non-normal distribution measurement data is expressed by a median (quartile spacing), and the comparison among groups is expressed by a rank sum test.P<A difference of 0.05 is statistically significant.

3. Analysis of Experimental results

1. Comparison before and after administration

Body weight before and after drug administration (71.36)vs 69.93 kg）、 BMI（25.32 vs 24.83 kg/m2 ）、FBG（7.80 vs 7.10 mmol/L）、GSP（291.00 vs275.00 umol/L) data statistically significant (P<0.05 Has comparability, waist-hip ratio (0.94)vs 0.93）、SBP（124 vs 118 mmHg）、DBP（77 vs75 mmHg）、TC（4.66 vs4.79 mmol/L）、TG（1.23 vs 1.28 mmol/L）、HDL（1.33 vs1.36 mmol/L）、LDL（2.43 vs2.59 mmol/L) etc. have no statistical significanceP>0.05 Comparison table of each index before and after treatment, as follows:

2. changes in ocular flora before and after administration

(1) Variation in α diversity: the curve of the accumulation of species, the abscissa is the sample size, and the ordinate is the number of OTUs, i.e. the number of species, the curve becomes gradually flat as the sample size increases, which indicates that the new species increase without significant change when the sample size is increased again, as shown in fig. 12. ACE (159.63) of ocular surface flora in T2DM patients before intervention (pre-E) and after intervention (post-E)vs 173.05）、Chao （156.35 vs175.67 And Shannon (3.39)vs3.81 The diversity index tends to increase after intervention, with the Shannon index being significantly higher after intervention than before intervention (b)p= 0.022), simpson index after intervention was significantly lower than before intervention (0.08)vs 0.06, p= 0.043), pre-E: pre-intervention, as shown in fig. 13; post-E Dry prognosis. These results indicate an increased alpha diversity of ocular surface flora after the intervention compared to before the intervention.

(2) β diversity change: PCoA analysis based on Weighted-UniFrac distance revealed that the ocular surface samples before and after intervention tended to cluster to different positions in the figure, as shown in FIG. 14, pre-E: before intervention; post-E is dry prognosis; adonis analysis based on Weighted-UniFrac distance found that there was a significant difference in ocular surface flora before and after intervention as a whole (S) ((R))P= 0.009); these results indicate that the ocular surface flora changes significantly overall after the intervention.

(3) Analysis of ocular surface flora species composition: phyla horizontal ocular surface flora species analysis shows that ocular surface flora is in proteobacteria before and after intervention: (Proteobacteria) (Geotrichum) and (D)Firmicutes) Actinomycetes door (Actinobacteriota) And Bacteroides (A), (B)Bacteroidota) As shown in fig. 15, the phylum of dominant bacteria; on the genus level, intervention in the anterior ocular surface flora with Acinetobacter (A)Acinetobacter) Ditz for genus of bacteria (A), (B)Dietzia) Halomonas genus (A), (B) and (C)Halomonas) Bacteroides genus (A), (B)Bacteroides) AndAliidiomarinathe genus is dominant, and the ocular surface flora after intervention is selected from Acinetobacter, bacteroides, dietzia,SubdoligranulumCorynebacterium, corynebacterium (II)Corynebacterium) And Halomonas are dominant genera, as shown in FIG. 16, pre-E: pre-intervention; post-E Dry prognosis.

And selecting the dominant bacterial group with obvious difference in ocular surface flora before and after intervention by adopting Mann-Whitney U test. At the phylum level, proteobacteria (50.72% vs 29.07%, p = 0.002) was found to be significantly higher before intervention than after intervention, while firmicutes (22.24% vs 37.36%, p = 0.009) and bacteroidetes (8.02% vs 14.41%, p = 0.024) were found to be significantly higher after intervention than before intervention, as shown in fig. 17; at the genus level, the genera acinetobacter (20.26% vs 9.31%, p = 0.005), waterborne (endobacter) (1.51% vs 0.53%, p = 0.034), serratia (Serratia) (0.65% vs 0.09%, p = 0.018), elizabeth (elibethkina) (0.47% vs 0.00%, p = 0.010), stenotrophomonas (Stenotrophomonas) (0.41% vs 0.06, p = 0.011) and the like were found to be significantly higher before intervention than after intervention, bacteroides (5.15% vs 10.56%, p = 0.014), subdolilolum (2.69% vs 5.90%, p = 0.006), coprobacterium (Faecalibacterium) (2.18% vs 4.08%, p = 0.010%, coprolimus) (1.01% vs 0.1.010%, rupesinus) (1.01%, rupess = 0.49%, p = 0.01%, rupess) (0.01%, rupess 0.49%, p = 0.01%, rupess) (1.01%, rupess 1.01%, p = 0.49%, p = 0.31.01%, p = 0.31%, p.011), and the like were found to be significantly higher before intervention

Coorabacter (Phascolarbacillus) (0.24% vs 0.80%, p = 0.011),

Parabacteroides (Parabacteroides) (0.23%; vs0.69%; p = 0.009); and,

Eubacterium coprostanoligenes_group（0.25% vs 0.48%, p=0.021）、

Parasxate (parasitella) (0.12% vs 0.45%, p = 0.002), eubacterium _ sirauum _ group (0.14% vs 0.36%, p = 0.039), lachnoclostrium (0.14% vs 0.32%, p = 0.039), butyricicccacea _ UCG-008 (0.09 vs 0.33, p = 0.031), parapherzia (paracretevolla) (0.09% vs 0.28%, p = 0.018), odoribacter (0.07 vs 0.24, p = 0.004), etc., were significantly higher after intervention than before intervention, pre-E: before intervention as shown in fig. 18; post-E Dry prognosis. * The numbers indicate that p values before and after intervention were significant by statistical tests, indicating p <0.05, p <0.01, p <0.001.

Based on LEfSe analysis, bacterial genera were selected that had significant differences in ocular surface flora before and after intervention. It was found that Bacteroides, subdoligranum, acetobacter, bacteroides (Oscilobacter), arthrobacter, choriophilus (Bilophila), parapropterium, odoribacterium, lachnospiraceae _ ND3007_ group, dorea, negatibacter, barnesiella, parasaxatilis, corarobacter, eubacterium _ silaum _ group, butyricoccaceae _ UCG _008, ruminococcus, lachnoclostrium, parabacteroides, ralstonia, and Eubacterium _ coprostagenigenes _ group, etc. were significantly higher after intervention than before intervention; before intervention, the genera Acinetobacter, enterobacter _ unclassified, niveispirillum, aquifex, blastomonas, wenyinglla (Veillonella), marseillea (Massilia), stenotrophomonas, and Serratia are significantly higher than after intervention, as shown in FIG. 19, pre-E: before intervention; post-E Dry prognosis.

Using random-forest analysis, 41 OTUs were found to differ in ocular surface flora before and after intervention, with 24 OTUs after intervention higher than before intervention belonging to lachnoclostrium, acinetobacter, brevibacillus (Brevibacillus), negatibacter, cholephagophilus, lachnospiraceae _ ND3007_ group, odonibacter, paraplasma, ralstonia, cladia, butyriciccicaceae _ UCG-008, spirillum (lachnoira), paracastemiella, dorea _ NK4a136_ group, corynebacterium, ruminococcus, bacteroides, subdoligranolaum, subdoligranoglucinum and coprobacillus, and the like; the remaining 17 OTUs, which were higher than the dry prognosis before intervention, were of the genus Marseillella, aeromonas (Aeromonas), prevotella (Prevotella), wenywort, blastomonas, granulesteella (Granulatella), acinetobacter, stenotrophomonas, serratia, streptococcus (Streptococcus), allorhizobium _ Neorhizobium _ Pararhizobium _ Rhizobium, dietzia, and the like, respectively, as shown in FIG. 20, pre-E: before intervention; post-E Dry prognosis.

(4) Functional prediction analysis: the sequencing data of the ocular surface flora 16S is based on a KEGG pathway database to predict the function of Tax4Fun2, and then a metabolic pathway (L3 level) with obvious difference in the ocular surface flora before and after intervention is selected through LEfSe analysis, wherein the selected differential metabolic pathway in the graph meets the condition that the P value is obvious and LDA is more than or equal to 3.0. Following intervention, the pathways for amino acid Biosynthesis (Biosynthesis of amino acids), starch and sucrose metabolism (Starch and sucrose metabolism), galactose metabolism (Galactose metabolism), other polysaccharide degradation (Other _ polysaccharide _ degradation), peptidoglycan Biosynthesis (Peptidoglycan _ Biosynthesis), homologus _ recombination, aminoacyl Biosynthesis (Aminoacyl tRNA Biosynthesis) are significantly elevated compared to those before intervention. Pathways for aromatic compound Degradation (Degradation of aromatic compounds), sulfur metabolism (Sulfur metabolism) were significantly reduced, as shown in FIG. 21, pre-E: before intervention; post-E Dry prognosis.

To summarize: the composition of normal ocular surface flora plays an important role in maintaining ocular health, ocular surface homeostasis, and preventing ocular infections. The core ocular surface flora accounts for 40% of the total ocular surface flora, wherein actinomycetes accounts for 53% of the core ocular surface flora, and secondly Proteobacteria accounts for 39% of the total ocular surface flora, and Thelephoraceae accounts for 8%, and healthy ocular surfaces can harmoniously interact with symbiotic flora, but ocular surface flora imbalance or transient flora increase can cause ocular diseases. Bacteria are considered to be the major cause of eye infections, which if left untreated, can lead to vision impairment and blindness. One survey found that about 33.07% of diabetic patients had both diabetic maculopathy and diabetic retinopathy, and 36.02% had both diabetic maculopathy and cataract. When Karimmab and colleagues used a culture method to compare conjunctival flora of diabetic patients and normal persons, the bacteria culture positive rate of the diabetic patients was found to be higher than that of non-diabetic patients, and the conjunctival culture positive rate of the persons with diabetic retinopathy was higher in the diabetic patients than that of the persons without retinopathy. The bilateral conjunctival culture positive rate of the proliferative diabetic retinopathy patient is obviously higher than that of the patient without retinopathy and non-proliferative diabetic retinopathy.

Experiments of the invention show that compared with the method before intervention, the relative abundance of acinetobacter and serratia after the intervention of canagliflozin is obviously reduced. Ham et al found that the abundance of acinetobacter on the ocular surface was significantly higher in diabetic patients compared to normal. Talreja et al, injected Acinetobacter baumannii into mouse eyes, found that the expressions of Interleukin-1 beta (Interleukin-1 beta, IL-1 beta), interleukin-6 (Interleukin-6, IL-6) and Tumor necrosis factor-alpha (TNF-alpha) in retina were significantly increased, and further showed that retinal cell death was increased by histology and cell death (TUNEL marker), thereby causing retinal damage and resulting loss of vision. Serratia marcescens, a rare but highly toxic cause of endophthalmitis, is an opportunistic pathogen for ocular infections. Endogenous endophthalmitis accounts for 5-10% of endophthalmitis cases, and diabetes is one of the risk factors, and can occur when microorganisms in the blood enter the eye, cross the blood retinal barrier, and infect ocular tissues. Animal experiment research by Zhou et al shows that Serratia can induce cornea inflammation by activating TLR4/MD-2/MyD88 and IL-1R1/MyD88 channels. The ocular surface flora participates in the occurrence and development of ocular diseases caused by T2DM, and the canagliflozin can reduce the risk of T2DM related ocular diseases by changing the ocular surface flora.

The experiment of the invention shows that: bacteroides were significantly elevated after dry prognosis. Qu et al found through in vivo and in vitro experiments that Bacteroides could reduce systemic inflammatory response by inducing the production of Short Chain Fatty Acids (SCFAs) and Interleukin-10 (IL-10). Currently, bacteroides are widely recognized as a beneficial group of bacteria that reduce inflammation by modulating lymphocyte and cytokine expression and controlling metabolism. Chronic inflammation and oxidative stress are considered as key components of the pathogenesis of diabetic retinopathy, and hyperglycemia causes local inflammation, mitochondrial dysfunction, microvascular dysfunction and apoptosis by inducing excess Reactive Oxygen Species (ROS) production, resulting in an increased incidence of diabetic retinopathy. ROS promote the production and activation of Nuclear factor-kappa beta (NF-kappa B), which in turn translocates to the nucleus and promotes the expression of inflammatory cytokines [ ], such as IL-1. Beta. And IL-6. Canagliflozin can activate AMP-activated protein kinase, inhibit secretion of IL-1 beta, monocyte chemotactic protein-1 (MCP-1) and IL-6, and reduce the degree of arteriosclerosis [ ]. As one study showed, canagliflozin treatment decreased levels of TNF receptor 1 (TNFR 1), IL-6, matrix metalloproteinase 7 (MMP-7), and Fibronectin 1 (Fibronectin 1, FN 1), indicating that canagliflozin has a reversing inflammatory effect [ ]. Furthermore, chen et al found by immunofluorescent staining that SGLT2, glucose transporter type 1 (GLUT 1) and glucose transporter type 5 (GLUT 5) levels in diabetic Lens Epithelial Cells (LECs) were significantly elevated compared to non-diabetic patients, and further studied that administration of daragliflozin treatment decreased SGLT2, GLUT1 and GLUT5 levels in sections of Lens epithelium, thereby down-regulating factors such as advanced glycation end products and phagocyte-type nicotinamide adenine dinucleotide phosphate oxidase, preventing ROS accumulation, and protecting LECs. Therefore, canagliflozin can reduce the inflammatory response and reduce the occurrence and development of eye diseases by increasing the flora which can down-regulate proinflammatory cytokines.

Based on functional predictions of KEGG, amino acid biosynthesis was found to be significantly enriched following canagliflozin treatment. In recent years, amino acids have been found to reduce inflammatory responses, maintain ocular surface homeostasis, and possibly ameliorate ocular surface diseases due to various causes. Wherein proline transport and utilization play an important role in maintaining retinal metabolism and health, and the retinal pigment epithelium can utilize proline to promote mitochondrial metabolism, synthesize amino acids, construct extracellular matrix, and resist oxidative stress. Dietary proline improves visual function in a mouse model of retinal pigment epithelium-specific oxidative damage ]. Thus, after administration of canagliflozin treatment, the ocular surface flora can reduce the inflammatory response by producing amino acids, thereby reducing the occurrence and development of ocular diseases.

In conclusion, the canagliflozin can regulate the structure of ocular surface flora of T2DM patients, reduce the ocular surface opportunistic flora and increase the protective flora, and the changed ocular surface flora can reduce inflammatory reaction by increasing amino acid biosynthesis.

Claims (4)

1. The new application of canagliflozin in reducing blood sugar of a type 2 diabetic patient is characterized in that: the new application is to regulate the intestinal flora of type 2 diabetes patients.

2. The canagliflozin of claim 1 can be used for a new application in reducing blood glucose in a type 2 diabetic patient, wherein: the new application is that the composition of intestinal flora is changed while FBG, GSP and body weight of a T2DM patient are reduced by using the canagliflozin treatment; at the phylum level, actinomycetemcomita is significantly lower after intervention than before intervention, while bacteroidetes is increased, at the genus level, drospirenone is significantly higher after intervention than before intervention, bifidobacterium, colepsy are reduced.

3. The new application of canagliflozin in reducing blood sugar of a type 2 diabetic patient is characterized in that: the new application is to regulate the ocular surface flora of type 2 diabetes patients.

4. The new use of canagliflozin in the treatment of glycemia in type 2 diabetic patients, in accordance with claim 3, characterized in that: the new application is that the canagliflozin reduces the opportunistic pathogenic flora of the ocular surface and increases the protective flora, and the changed ocular surface flora reduces the inflammatory response by increasing the biosynthesis of amino acid, thereby reducing the occurrence of ocular diseases.

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