Bifidobacterium lactis Probio-M8 ameliorated the symptoms of type 2 diabetes mellitus mice by changing ileum FXR-CYP7A1

Ye Chen; Yaxin Zhao; Xin Shen; Feiyan Zhao; Jinxin Qi; Zhi Zhong; Dongmei Li

doi:10.1515/med-2022-0576

Article Open Access

Bifidobacterium lactis Probio-M8 ameliorated the symptoms of type 2 diabetes mellitus mice by changing ileum FXR-CYP7A1

Ye Chen , Yaxin Zhao , Xin Shen , Feiyan Zhao , Jinxin Qi , Zhi Zhong and Dongmei Li

Published/Copyright: December 15, 2022

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From the journal Open Medicine Volume 17 Issue 1

Abstract

The aim of this study was to investigate the effect of Bifidobacterium lactis Probio-M8 on glucolipid metabolism and gut microbiota (GM) composition in type 2 diabetes mellitus (T2DM) mice. The glucolipid metabolic profiles were analyzed. The 16S rRNA gene sequencing was employed to investigate GM. The levels of farnesyl X receptor (FXR) and cytochrome p450 7A1 (CYP7A1) were detected by quantitative polymerase chain reaction and western blot assays. The total bile acids (TBAs), ceramide (CE), glucagon-like peptide-1 (GLP-1), and fibroblast growth factor (FGF)-15 were also detected. The morphological features of liver and pancreas were also analyzed. Compared with the model group, Probio-M8 restored body weight, food intake and water intake, as well as improved hyperglycemia symptoms, serum glucolipid parameters, and the composition of intestinal microbes in T2DM diabetic mice. Moreover, the reduced level of FXR and the increased level of CYP7A1 in T2DM mice were reversed by Probio-M8 treatment. The increased levels of TBA and CE and the reduced levels of GLP-1 and FGF-15 in T2DM mice were altered after Probio-M8 stimulation. Besides, the altered morphology of liver and ileum in T2DM mice was alleviated by Probio-M8 treatment. Taken together, we suggested that the symptoms of T2DM could be ameliorated by Probio-M8 in T2DM mice.

Keywords: Probio-M8; type 2 diabetes mellitus; FXR-CYP7A1; glucolipid metabolism; gut microbiota

1 Introduction

Type 2 diabetes mellitus (T2DM) is a disorder of glucose metabolism caused by a combination of genetic and environmental factors. Obesity and insulin resistance (IR) are risk factors for the development of T2DM. In recent years, a large number of studies have shown that gut microbiota (GM), which is an important environmental factor, is closely related to the development of obesity, IR, T2DM, and other metabolic diseases [1].

Recent studies demonstrated that bile acids (BAs) and farnesyl X receptor (FXR) play a critical role in the glucose and lipid metabolic homeostasis. BAs are produced in the liver via the hydroxylation of cholesterol, while cytochrome p450 7A1 (CYP7A1) is the rate-limiting enzyme in major step. In gut, some microbial species mediate the depolymerization of BAs and can further metabolize secondary Bas, which can activate FXR [2]. FXR is most plentifully expressed in the intestine and liver. Activation of FXR in enterocytes leads to the upregulation of fibroblast growth factor (FGF)-19 in humans and orthologous FGF-15 in mice [3,4]. Furthermore, inhibition of intestinal FXR has been found to reduce ceramide (CE) secretion, which could decrease the hepatic glycogen xenobiogenesis [5], and promote the glucagon-like peptide-1 (GLP-1) secretion, which furtherly increase insulin secretion after meals therefore alleviate IR [6].

Probiotics are living microorganisms that can bring benefits to the host at an appropriate dose. Some evidence-based studies displayed that some probiotics show benefit for glucose metabolism in T2DM humans [7,8]. Preliminary researches revealed that oral probiotics can improve intestinal flora disorders and reduce blood glucose, lipids, and IR in animal models [9,10], whereas the mechanism is still not fully understood [11]. In T2DM-related studies, Chen et al. found that the compound probiotics containing Probio-M8 can effectively enhance the hypoglycemic effect of metformin in patients with T2DM, accompanied by a significant decrease in fasting blood glucose, glycosylated hemoglobin, and blood uric acid, and increase beneficial bacteria, such as Bifidobacterium and Eubacterium hallii. However, no single strain of Probio-M8 has been studied on glucose metabolism [12].

This study aims to investigate the glycolipid metabolism profiles, intestinal microbiome, and serum and tissue metabolites for understanding the effect and mechanism of Bifidobacterium lactis Probio-M8 on T2DM.

2 Materials and methods

2.1 Preparation of lactic acid bacteria suspension

B. lactis M8 (Probio-M8) (batch N0: 20201117011) used in our study was obtained from JinHua YinHe Biological Technology Co. (Zhejiang, China; prepared under ISO9001). Before intragastric administration, the Probio-M8 was suspended in normal saline with 4 × 10⁹ CFU/ml.

2.2 Animals and experimental design

All procedures involving animals were approved by the animal care review committee of Inner Mongolia Agricultural University and adhered to the institutional animal care committee guidelines.

Male C57BL/6J mice (5 weeks old, 140–160 g) were subjected to a standard diet and fresh water acclimation for 1 week at a temperature of 23 ± 1°C, a humidity of 54 ± 2%, and light/dark cycle of 12 h. All mice were randomly divided into three groups: control group fed a normal rat chow diet; model group fed a high-fat diet; and probiotic treatment group fed the same diet as the model groups. After 2 weeks, the mice in model and probiotic treatment groups were intraperitoneally injected with streptozocin (STZ), while the control group mice were intraperitoneally injected with saline solution. Thereafter, the mice in the probiotic group were intragastric with Probio-M8 (4.0 × 10⁹ CFU/mice per day, suspended in normal saline with 4 × 10⁹ CFU/ml) for 8 weeks, while the mice in the model groups were intragastric with saline (1 ml/mice per day) for 8 weeks.

2.3 Collection of blood and tissue sample

At the end of the experiments, blood samples were collected and centrifuged to obtain blood serum for following detection. Then, the liver, ileum, and pancreas tissues were obtained and divided into two parts, one of which was washed with pre-cooling saline and placed in formalin and the other was used for protein extraction. At the same time, a sample of mice feces was collected and stored in a refrigerator at −80°C for later use.

2.4 Fasting blood glucose (FBG) and oral glucose tolerance

After the probiotic treatment, the mice were fasted for 12 h to measure the fasting blood glucose, and then, the oral glucose tolerance tests (OGTT) were performed. Mice were fasted for 12 h and then administered glucose 2 g/kg body weight orally. Blood glucose levels were measured at 0, 30, 60, 90, and 120 min using a portable contour blood glucose monitor. Finally, the oral glucose tolerance curve was plotted, the area under the curve (AUC) was calculated, and the differences between the groups were compared. Glucose AUC = 1/4 × fasting blood glucose value + 30 min blood glucose value + 60 min blood glucose value + 90 min blood glucose value.

2.5 Detection of blood biochemical indexes

Blood samples of all mice were collected and mouse triglyceride (TG) ELISA kit (catalog no. BH8381; BOYAO, China), mouse total cholesterol (TC) ELISA kit (catalog no. ab285242; Abcam, USA), mouse high-density lipoprotein cholesterol (HDL-C) ELISA kit (catalog no. CSB-E12874m; CUSABIO, China), and low-density lipoprotein (LDL) cholesterol ELISA kit (catalog no. CSB-EQ027860MO; CUSABIO) were applied to detect the serum concentrations of TG, TC, HDL-C, and LDL, respectively. Moreover, the plasma levels of CE (catalog no. ZC-38647; ZCIBIO, China), total bile acid (total bile acid [TBA], catalog no. ab239702; Abcam), glycated albumin (catalog no. MBS029310; MyBioSource, USA), GLP-1 (catalog no. CSB-E08118m; CUSABIO), insulin (catalog no. ab277390; Abcam), and FGF-15 (catalog no. CSB-EL522052MO; CUSABIO) were quantified by the corresponding ELISA kit. The steps were operated according to the instructions of the kit.

2.6 Extraction of fecal metagenomic DNA

Take 0.5 g stool sample, extract metagenomic NDA using QIAamp kit, 1% agarose gel electrophoresis, Nanopore drop, and Qubit^® 2.0 fluorometer to evaluate the integrity and concentration of the extracted genomic DNA, and perform polymerase chain reaction (PCR) on qualified DNA amplification.

2.7 PCR amplification, library building, and sequencing

KAPA HiFi HotStart ReadyMix PCR kit (America) and primers with different barcodes were used for 16S rRNA amplification. The amplification system is: KAPA HiFi HotStart ReadyMix 25 μl; forward and reverse primers (10 μmol/l) each 1.2 μl; DNA template 100 ng; and dd H₂O up to 50 μl. PCR amplification program is: (1) pre-denaturation 98°C, 3 min; (2) denaturation 98°C, 20 s, annealing 62°C, 15 s, extension 72°C, 45 s, 26 cycles; and (3) terminal extension 72°C, 90 s, 4°C termination. Amplify 16S rRNA according to the amplification system and amplification conditions and mix and purify the PCR products that meet the library building conditions. Pacific Biosciences SMRTbell™ Template Prep Kit 1.0 kit (Pacific Biosciences Corporation) was used to construct the library of the purified mixed sample, DNA/Polymerase Bingding Kit P6 v2 (Pacific Biosciences Corporation) was used to add sequencing primers and polymerase to the constructed library, and then, Pacbio SMRT RS Ⅱ was used to sequence.

2.8 Bioinformatics analysis

RS_ReadsOfinsert.1 was used to perform the quality control on the measured original sequence. The specific conditions are: the minimum number of cycles is 5, the minimum prediction accuracy is 90%, the minimum insert is 1400, and the maximum insert is 1800. Use the QIIME [13] (V1.7.0) platform to perform bioinformatics analysis of high-quality sequences after quality control. First, use PyNAST [14] to align the sequences, perform UCLUST [15] merge, divide the classification operation unit (OTU) at 97 and 100% similarity, select one from each out, and use SLIVA [16], greengene [17], and RDP for homology comparison to determine the classification Academic status.

2.9 Total RNA isolation and real-time quantitative PCR (qPCR)

Total RNA was extracted from the liver and ileum by using the RNApure kit (Biolab, Beijing, China) and reverse transcriptase SuperScript III was exploited to transcript cDNA. The PCR condition was 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s and a final extension of 72°C for 10 min. The GAPDH was regarded as an internal control for FXR and CYP7A1 detection. The relation expression was calculated by the 2^−ΔΔCt method.

2.10 Western blotting

The liver and ileum tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer, the protein concentration was analyzed by using a BCA protein assay kit (Pierce, Rockford, IL, USA). Then, equal amounts of protein extracts (25 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. After blocking in 5% nonfat milk for 2 h, the membranes were incubated with the indicated primary antibodies overnight for 4°C and the corresponding secondary antibody for 2 h at ambient temperature. Finally, the signals were detected and analyzed by ImageJ software.

2.11 Oil-red O staining

For oil-red O staining, the liver tissues and epididymal adipose tissues were cut into 8 µm thick sections and fixed with 4% paraformaldehyde. Next, the sections were washed with phosphate buffer saline and then stained with hematoxylin reagent. Subsequently, the sections were stained with oil-red O staining reagent after incubation in 60% isopropanol for 10 min. Finally, the sections were rinsed with running water and observed under a microscope.

2.12 Histomorphological analysis

At the end of the experiment, the liver and pancreas tissues were dissected, fixed with 4% paraformaldehyde solution, and embedded in paraffin. Then, 5 µm thick samples were prepared and stained with hematoxylin–eosin (HE). Finally, the samples were observed and photographed under an optical microscope.

2.13 Statistical analysis

All experiments were repeated for three times. The experimental data were displayed as mean ± standard deviation (SD) and analyzed by using GraphPad Prism 6. The differences between each group of data were analyzed by one-way analysis of variance followed by Bonferroni’s post hoc test. P value <0.05 was deemed as statistically significant. R (v3.3.2) was used to analyze α diversity and β diversity, Wilcoxon rank sum test was used to calculate the difference between each group, Spearman correlation coefficient is calculated to analyze the correlation between microbes and indicators, and cytoscape (v3.5) was used to analyze a network diagram.

Ethics: All procedures involving animals were approved by the animal care review committee of Inner Mongolia Agricultural University and adhered to the institutional animal care committee guidelines.

3 Results

3.1 Probio-M8 improved hyperglycemia symptoms and glycolipid profiles in T2DM mice

The body weight, food intake, and water consumption of mice under different stimulations are displayed in Tables 1–3. The body weight of mice in the model group reduced and showed an obvious difference with the control group after STZ injection 4 weeks. However, the use of Probio-M8 slowed the weight loss of mice in model group (Table 1). Moreover, the food intake and water consumption of mice in model group were increased, which exhibited a significant difference with the control group after STZ injection, while the administration of Probio-M8 reduced the intake of food and the consumption of water and showed an obvious difference with the model group (Tables 2 and 3).

Table 1

Effects of Probio-M8 strains on weight (g/day) of T2D mice

Group/week	1	2	3	4	5	6	7	8
Control	23.85 ± 1.57	26.25 ± 1.77	28.09 ± 2.11	29.60 ± 2.41	31.43 ± 2.04	32.80 ± 2.49	34.35 ± 2.70	36.00 ± 2.80
Model	27.07 ± 1.28	26.50 ± 1.49	25.10 ± 1.59	23.40 ± 1.58*	22.12 ± 1.54*	21.00 ± 1.75*	20.04 ± 1.19*	18.85 ± 1.10*
Probiotics	26.70 ± 1.28	26.40 ± 1.50	25.39 ± 1.39	24.81 ± 1.30	24.68 ± 1.40	24.47 ± 1.51	23.95 ± 1.59	23.80 ± 1.73^#

^* P < 0.05 vs the control group.

^# P < 0.05 vs the model group.

Table 2

Effects of Probio-M8 strains on food intake (g/day) of T2D mice

Group/week	1	2	3	4	5	6	7	8
Control	42.92 ± 3.65	43.67 ± 3.84	43.70 ± 3.59	42.33 ± 2.75	42.82 ± 2.91	43.60 ± 3.62	42.67 ± 2.14	42.55 ± 3.75
Model	54.57 ± 4.19*	55.32 ± 4.45*	57.88 ± 2.59*	54.02 ± 3.22*	56.63 ± 1.15*	57.02 ± 2.14*	55.25 ± 2.63*	55.47 ± 2.12*
Probiotics	51.65 ± 3.63	50.80 ± 2.63	49.88 ± 4.20^#	47.88 ± 2.40^#	48.72 ± 3.54^#	49.12 ± 1.66^#	50.47 ± 3.03^#	49.25 ± 2.44^#

*P < 0.05 vs the control group.

^# P < 0.05 vs the model group.

Table 3

Effects of Probio-M8 strains on water consumption (g/day) of T2D mice

Group/week	1	2	3	4	5	6	7	8
Control	29.60 ± 3.37	26.18 ± 2.84	26.45 ± 3.63	29.23 ± 5.62	28.02 ± 2.00	30.27 ± 3.54	28.23 ± 2.88	26.97 ± 2.69
Model	42.27 ± 5.07*	43.10 ± 2.75*	43.70 ± 2.66*	43.92 ± 1.79*	45.10 ± 2.93*	44.07 ± 2.05*	42.68 ± 4.26*	44.68 ± 3.47*
Probiotics	41.70 ± 3.59	41.48 ± 1.51	40.50 ± 3.19	40.27 ± 2.08	39.95 ± 1.77^#	39.22 ± 1.82^#	38.62 ± 0.89^#	38.18 ± 3.02^#

^* P < 0.05 vs the control group.

^# P < 0.05 vs the model group.

Results from OGTT showed that the AUC of the model group was obviously larger than that of the control group, which indicated that the blood glucose tolerance of the model group was decreased obviously. However, compared with the model group, the T2DM mice stimulated by Probio-M8 showed lower AUC (Figure 1a) and higher blood glucose tolerance. As presented in Figure 1b, the blood glucose level in the model group was remarkably increased compared with the control group, while Probio-M8 treatment significantly reduced the blood glucose level in the model group.

Figure 1

AUC and several blood biochemical indexes were analyzed after the administration of Probio-M8 in T2DM mice. (a) The AUC in the control, model, and probiotic groups was analyzed. (b and c) The increase level of glucose and decreased level of HDL in T2DM mice were suppressed by the addition of Probio-M8. (d–f) The increased levels of LDL, TG, and TC in T2DM mice were eliminated by the addition of Probio-M8. (g) The increase level of GA in T2DM mice was eliminated by the addition of Probio-M8. (h) The reduced level of insulin in T2DM mice was eliminated by the addition of Probio-M8. **P < 0.01 vs control, ^## P < 0.01 vs model.

Analysis from commercial kit showed that HDL level was reduced in the model group, and the levels of LDL, TG, and TC were increased in the model group compared with the control group; however, Probio-M8 treatment remarkably elevated the HDL level and reduced the levels of LDL, TG, and TC in the model group (Figure 1c–f).

Data from Figure 1g and h displayed the comparative data of GA and insulin. The model mice showed an obvious increase in GA, along with lower level of insulin compared with the control group. However, the treatment with Probio-M8 provided a significant reduction of GA, as well as a marked increase in the level of insulin.

3.2 Probio-M8 modified the key gut bacteria in T2DM mice

The Shannon index and Simpson index were used to evaluate the species diversity of intestinal microbes. After the experiment, the α diversity (Shannon index and Simpson index) of the control group, the model group, and the probiotic group showed no significant change, but the intestinal microbes of the model group mice α diversity showed an upward trend, while the probiotic group and control group α diversity is relatively consistent (Figure 2). The principal component analysis (PCA) results show that there was a significant difference between the probiotic group and the control group (P < 0.05). The Wilcox test was used to calculate the differences between the three groups of bacteria, and they were defined as differential bacteria. In our study, a total of four different species were screened, namely (Figure 3), Flintibacter butyricus, Bacteroides nordii, Parabacteroides distasoni, and Oscillibacter valericigenes.

Figure 2

Microbial structure differences. (a) Shannon index of the control group, the model group, and the probiotic group. (b) Simpson index of the control group, the model group, and the probiotic group. (c) PCA of the control group, the model group, and the probiotic group. (d) Calculate P values and R values between groups; a P value indicated whether there is a statistical difference between the groups, and R (v3.3.2) was used to analyze α diversity and β diversity. P value of <0.05 was considered statistically significant.

Figure 3

Significant differential species among the control group, the model group, and the probiotic group. P value of <0.05 was considered statistically significant.

Among them, B. nordii increased significantly in the model group (P < 0.05), while the probiotic group and the control group were at the same level. Compared with the probiotic group and the control group, Pseudoflavonifractor capillosus and O. valericigenes decreased significantly in the model group. In this study, the correlation coefficient of spearman in R software was used to calculate the correlation value and P value between marker bacteria and indicators and screened out those with significant correlation according to r < −0.5 and r > 0.5 (P < 0.05) to construct the correlation network diagram (Figure 4). We found that B. nordii, F. butyricus, O. valericigenes, P. capillosus, and Vampirovibrio chlorellavorus have a strong correlation with blood glucose and blood lipids. B. nordii is positively correlated with TG, CE, TBA, and blood sugar and negatively correlated with HDL and insulin. Other different bacteria also have a certain correlation with blood glucose and other indicators. Therefore, it shows that the intestinal microbes are closely related to the occurrence of T2DM, and the intestinal microbes can regulate the host’s glycolipid metabolism.

Figure 4

The correlation between indicators such as blood glucose metabolism, blood lipids, and different species. Red represents positive correlation, blue represents negative correlation, and the thickness of the line represents the strength of the correlation.

3.3 FXR-CYP7A1 axis changed in the function of Probio-M8 in mice with T2DM

FXR is an important molecular mediator of diverse metabolic processes, such as modulation of BAs and lipid and glucose homeostasis. Therefore, FXR expression in ileum tissues was examined in our study. Results from Figure 5a and b showed that the mRNA and protein levels of FXR in ileum were obviously reduced in T2DM mice, whereas the addition of Probio-M8 significantly increased the FXR level. In hepatocytes, the classical pathway is the main pathway of BAs’ synthesis, and CYP7A1 is the rate-limiting enzyme in the classical pathway [18]. Therefore, qPCR and western blot were used to detect CYP7A1 levels. CYP7A1 expression in liver was opposite to that of the FXR in T2DM mice, and the addition of Probio-M8 reduced CYP7A1 expression in liver (Figure 5c and d). We also determined the TBA level and found that the model mice showed an obvious increase in TBA, whereas the treatment with Probio-M8 provided a significant reduction of TBA comparing with the T2DM mice (Figure 5e).

Figure 5

The levels of FXR and CYP7A1 in liver and ileum were analyzed by qPCR and western blot assays, and the blood biochemical indexes were detected after the addition of Probio-M8 in T2DM mice. (a and b) The level of FXR was reduced in ileum of T2DM mice, whereas the addition of Probio-M8 altered the condition. (c and d) The level of CYP7A1 in liver of T2DM mice was increased; however, the addition of Probio-M8 eliminated the phenomenon. (e) The increase level of TBA in T2DM mice was eliminated by the addition of Probio-M8. (f) The increase level of CE in T2DM mice was eliminated by the addition of Probio-M8. (g and h) The reduced levels of GLP-1 and FGF-15 in T2DM mice were eliminated by the addition of Probio-M8. **P < 0.01 vs control, ^## P < 0.01 vs model.

3.4 CE, GLP-1, and FGF-15 were improved in T2DM mice by Probio-M8

Data from Figure 5f–h displayed the comparative data of CE, GLP-1, and FGF-15 in different groups. As we expected, the model mice showed an obvious increase in CE, along with lower levels of GLP-1 and FGF-15 when compared with the control group. However, the treatment with Probio-M8 provided a significant reduction in the release of CE, as well as a marked increase in the levels of serum GLP-1 and FGF-15.

3.5 Probio-M8 protected against histological damage in mice with T2DM

To identify improvement in the syndromes of T2DM with Probio-M8, we carried out histological analysis of the liver and pancreas of the mice in the study. In the liver, the administration of Probio-M8 decreased hepatocyte edema and hepatic sinus congestion (Figure 6a). The structure of control group was normal, and the acinar cells were highly stained and closely arranged. Then, we observed significant differences in the pancreatic shape and number of islets between the control and model groups, indicating necrosis of acinar cells around the islets. However, Probio-M8 treatment partially restored islet cells and reduced β cells’ necrosis and vacuolation (Figure 6b).

Figure 6

HE staining of liver and pancreas and relationship between Probio-M8 administration and inflammatory changes in epididymal adipose tissue and liver. (a and b) After the addition of Probio-M8, the histological changes in liver and pancreas in T2DM mice were improved. (c) Oil-red O staining of the liver tissues was presented. (d) Oil-red O staining of the epididymal adipose tissues was presented.

Histological analysis revealed a large increase in the accumulation of fat in the liver of the model mice, which was alleviated by the administration of Probio-M8 (Figure 6c). Analysis from epididymal adipose tissue showed that significant macrophages infiltrated into adipose tissue and typical coronal structures were observed histologically in model mice; however, the above phenomena were changed after the addition of Probio-M8 (Figure 6d).

4 Discussion

Our work constructed a T2DM mouse model with a high-fat diet and STZ, which use Probio-M8 for an 8-week intervention, and found that Probio-M8 improved the relevant indicators of T2DM. Probio-M8 can improve the general symptoms of diabetes, such as polydipsia, polyphagia, and weight loss in T2DM mice. FBG reflects basal insulin secretion and blood glucose status under the stimulus of no glucose load, OGTT is designed to understand islet β-cell function and the body’s ability to regulate blood glucose under glucose load, and GA reflects the average blood glucose level in the past 2–3 weeks. Probio-M8 treatment can improve the levels of FBG, OGTT, and GA. T2DM is mainly caused by IR and insufficient insulin secretion. After the intervention of Probio-M8, the level of FINS in T2DM mice was improved, indicating that Probio-M8 can promote insulin secretion and protect islet function. Aberrant blood lipid profiles often occurred in patients with T2DM, for example, the total levels of TC, TG, and LDL in patients with T2DM were obviously higher than those in normal individuals [19]. Some blood glucose regulatory strains have been reported to improve blood lipid profiles in patients with T2DM [20]. In accordance with the published findings, our study found that Probio-M8 decreased TC and TG levels and improved HDL/LDL ratios in T2DM mice.

Intestinal microbes and its metabolites can affect the occurrence and development of metabolic diseases. Many studies reported the lower abundance of Bacteroides have found in the intestines of obese mice or diabetic mice [21]. In this study, compared with the control group, the Bacteroides phylum of the model group decreased, and after supplementing with Probio-M8, the content of Bacteroides tended to increase. Therefore, Probio-M8 can restore the content of Bacteroides in the intestine of diabetic mice, which is consistent with the results of previous studies [22]. In this study, compared with the model group, the probiotic group and the control group have the same content levels trend in O. valericigenes and P. capillosus. O. valericigenes and P. capillosus have the ability to produce butyric acid and acetic acid. Studies have shown that butyric acid produced by GM can improve the body’s insulin response. Butyric acid, propionic acid, and acetic acid are important components of SCFAs, and more and more studies have shown that SCFAs have beneficial effects on inflammation, insulin sensitivity, and blood glucose homeostasis [23]; therefore, these findings suggest that the increase in butyric acid-producing bacteria may be helpful in the treatment of T2DM [24]. It is worth noting that Parabacteroides distasonis is one of the core microbes of the human body and the abundance of P. distasonis is significantly negatively correlated with obesity, nonalcoholic fatty liver, diabetes, and other disease states, suggesting that it may play a positive regulatory role in glucose and lipid metabolism. However, in this study, compared with the model group and the control group, P. distasonis decreased in the probiotic group, which is inconsistent with the results of previous studies. The reason may be that this study is based on 16S rRNA for genome analysis, which has certain limitations in species identification. Follow-up studies can use metagenomics technology to reveal from deeper level changes in the structure of the microbes.

In this study, Probio-M8 can restore the changes in the structure of the intestinal microbes in T2DM mice. Combined with the analysis of clinical results, it is found that the changes in the composition of the intestinal microbes are related to the improvement of clinical results. In the intestine, intestinal microbes can change the bile acid pool and activate FXR to trigger the production and secretion of FGF-15 [20]. FGF-15/19 can play an insulin-like role in inhibiting gluconeogenesis, promoting the synthesis of glycogen and protein, reducing dietary intake, losing body weight, and improving insulin sensitivity and glucose tolerance [25,26]. Activation of intestinal FXR has been reported to reduce obesity and IR in mouse models of diseases [27]. Moreover, some published studies showed that probiotic treatment can improve glucose homeostasis by enhancing FXR signaling. In our study, the reduced FXR expression in ileum was inhibited after Probio-M8 treatment in T2DM mice. FXR has been found to bind to a response element located in the second intron of the FGF-15 gene to directly regulate its transcription [20]. Significantly, in our study, we observed that FGF-15 level was reduced in T2DM mice; however, Probio-M8 treatment inhibited the phenomenon. In the liver, FXR activation contributed to liver regeneration, as well as glucose, lipid, and cholesterol homeostasis [28,29]. Consistent with the above-published reports, activation of hepatic FXR is beneficial in the treatment of diabetes and nonalcoholic fatty liver disease [30]. A previous study showed that the expression and activity of CYP7A1 in FGF-15-KO mice increased, and BAs synthesis increased accordingly [20]. Moreover, previously published reports indicated that the feedback regulation of CYP7A1 by BAs is mediated by a nuclear receptor signaling cascade involving FXR [31]. Depletion of FXR in intestine disturbs FXR-mediated inhibition of CYP7A1, while depletion of FXR in liver does not [32]. Intestinal FXR activated by BAs downregulated CYP7A1 expression in liver indirectly by the intestinal FGF-15 synthesis and secretion [33]. Analogously, in our study, we observed that the expression of FXR in the ileum of T2DM mice was significantly reduced, and after treatment with Probiotic M8, the expression of FXR was reversed. At the same time, the expression of CYP7A1 in the liver was regulated by FXR, its expression in the liver was significantly increased in T2DM mice, while the increased CYP7A1 level was obviously suppressed after Probiotic M8 treatment in T2DM mice.

CE was reported to reduce muscle insulin sensitivity and stimulate reactive oxygen species production at mitochondrial level [20]. In our study, we observed that the increased level of CE in T2DM was reduced after Probio-M8 treatment.

5 Conclusions

In summary, our results showed that Probio-M8 can restore body weight, food intake, and water intake and improve blood lipids and glucose tolerance in T2DM mice. Probio-M8 can adjust the composition of BA by changing the structure of the intestinal microbes, increase the level of FXR in the ileum, trigger the production and secretion of the rodent ortholog FGF-15, reduce the level of CYP7A1 in the liver, and restore the morphology of the liver and pancreas. Therefore, the hypoglycemic mechanism of Probio-M8 on T2DM mice may be through the change in ileum FXR-CYP7A1, suggesting that Probio-M8 can be used as a potential probiotic for the treatment of T2DM.

Abbreviations

T2DM: type 2 diabetes mellitus
TBAs: total bile acids
CE: ceramide
GLP-1: glucagon-like peptide-1
FGF: fibroblast growth factor
IR: insulin resistance
GM: gut microbiota
FXR: farnesyl X receptor
CYP7A1: cytochrome p450 7A1
FGF19: fibroblast growth factor 19
Probio-M8: Bifidobacterium lactis M8
STZ: streptozocin
OGTT: oral glucose tolerance tests
AUC: area under the curve
TG: triglyceride
TC: total cholesterol
HDL-C: high-density lipoprotein cholesterol
LDL: low-density lipoprotein cholesterol
GA: glycated albumin
OTU: operation unit
RIPA: radioimmunoprecipitation assay
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
PVDF: polyvinylidene fluoride
HE: hematoxylin-eosin
SD: standard deviation
FBG: fasting blood-glucose.

Acknowledgments

None.

Funding information: This study was supported by Inner Mongolia Science and Technology Major Projects (2021ZD0014), Inner Mongolia Natural Science Foundation Project (2021LHMS08061), and Hospital fund project of Inner Mongolia People’s Hospital (2019YN02).
Author contributions: Ye Chen conceived the study, performed the experiments, analyzed the data, and wrote the article. Yaxin Zhao performed the experiments, analyzed the data, and wrote the article. Xin Shen performed the experiments and wrote the article. Feiyan Zhao analyzed the data and wrote the article. Jinxin Qi wrote the article. Zhi Zhong and Dongmei Li conceived the study and reviewed the article. All authors read and approved the final version.
Conflict of interest: The authors declare that there is no conflict of interest.
Data availability statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2022-03-24

Revised: 2022-09-02

Accepted: 2022-09-05

Published Online: 2022-12-15

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/med-2022-0576

Keywords for this article

Probio-M8; type 2 diabetes mellitus; FXR-CYP7A1; glucolipid metabolism; gut microbiota

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BY 4.0