Astragalus mongholicus polysaccharides alleviate insulin resistance through modulation of PI3K/AKT, TLR4/NF-kB signaling pathway and microbiota in rats with Type 2 Diabetes Mellitus

Abstract

Background and aim

Astragali Radix has been widely used in traditional Chinese medicine to treat diabetes and a variety of other diseases. This study aims to evaluate the alleviating effects and mechanisms of Astragalus mongholicus Polysaccharide (mAPS) against diet combined with streptozotocin (STZ)-induced Type 2 Diabetes Mellitus (T2DM).

Experimental procedure

T2DM rats were orally administrated either with 200 mg/kg mAPS or 300 mg/kg Metformin (MET) once daily for four weeks. Body weight and Fasting Blood Glucose (FBG) were detected every 6 days. Serum fasting insulin (FINS) was measured by ELISA and the homeostatic model assessment of insulin resistance (HOMA-IR) was calculated accordingly. Histological change was studied by Hematoxylin and eosin (HE) staining. 16S rDNA sequencing was used to detect the changes in gut microbiota.

Results and conclusion

Oral administration of mAPS significantly decreased body weight, FBG, and HOMA-IR in T2DM rats (p＜0.05). Moreover, HE staining showed that mAPS could alleviate histological distortion in the liver and pancreas. Treatment with mAPS elevated the hepatic levels of phosphatidylinositol-3 kinase (PI3K), phospho-protein kinase B (AKT), and glucose transporter type 4 (GLUT4), while reducing phospho-nuclear factor kappa-B (NF-κB), Toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) (p＜0.05). Furthermore, mAPS supplementation could reverse the ratio of Firmicutes/Bacteroidetes (F/B) and reduce the abundance of Clostridia and Proteobacteria (p＜0.05). These results indicate that mAPS have the potential to enhance insulin sensitivity in diabetic rats by modifying gut microbiota and controlling the hepatic glycolipid metabolism and inflammation.

Graphical abstract

Abbreviations

Akt

Protein kinase B

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

ELISA

Enzyme-linked immunosorbent assay

FBG

Fasting blood glucose

FINS

Fasting insulin

GLUT4

Glucose transporter type 4

HDL

High-density lipoprotein

HE

Hematoxylin and eosin

HOMA-IR

Homeostatic model assessment of insulin resistance

HSHFD

High-sugar and high-fat diet

IL-1β

Interleukin-1β

IL-6

Interleukin-6

IR

Insulin resistance

ISI

Insulin sensitivity index

LDL

Low-density lipoprotein

LPS

Lipopolysaccharide

mAPS

Astragalus mongholicus polysaccharides

MET

Metformin

NF-κB

Nuclear factor kappa-B

PCR

Polymerase chain reaction

PI3K

Phosphatidylinositol-3 kinase

STZ

Streptozotocin

TC

Total cholesterol

TG

Triglyceride

TNF-α

Tumor necrosis factor-α

TLR4

Toll-like receptor 4

T2DM

Type 2 diabetes mellitus

ZO-1

Zona Occludens 1

1. Introduction

Type 2 Diabetes mellitus (T2DM) represents a multifaceted disorder caused by insufficient insulin secretion and insulin resistance (IR).^1,2 As a typical metabolic disease, its occurrence and development not only lead to glycolipid metabolism disorder but also lead to the gut microbiota dysbiosis, which is often accompanied by low-grade inflammation.³ Based on the data provided by the International Diabetes Federation (IDF), the global diabetic populace exceeded 450 million (2017), and it is estimated to escalate to 629 million (2045).⁴ Therefore, the exploration of new agents with both hypoglycemic and gut-microbiota regulatory effects to treat T2DM is urgent.

The liver plays a critical role in the development of T2DM.⁵ PI3K/Akt signaling pathway regulates fundamental cellular functions in glucose metabolism.⁶ Activation of the PI3K/Akt signaling pathway can promote GLUT4 transfer to the cell plasma membrane, leading to improved glucose uptake.⁷

Gut microbiota are a complex community that is mainly populated by bacteria (>90 %).⁸ Gut microbiota dysbiosis contributes to the increased prevalence of T2DM and obesity.⁹ It has been demonstrated that the relative abundance of Proteobacteria was increased in diabetic patients,¹⁰ and there was an increase of the Firmicutes-to-Bacteroidetes ratio in the rats model of T2DM.¹¹ Modulation of gut microbiota has the potential to improve the treatment of T2DM.^12,13 Activation of the TLR4/NF-κB signaling pathway results in the synthesis and production of a variety of inflammatory cytokines.¹⁴ The activation of TLR4/NF-κB signaling pathway and low-grade inflammation are associated with gut microflora dysbiosis in T2DM.¹⁵

Astragali Radix (“Huangqi” in Chinese) has long been utilized in traditional Chinese medicine to treat diverse ailments such as fatty liver, enteropathy, cancer, diabetes, and cardiovascular disease.¹⁶ As a variant of Astragali Radix, Astragalus membranaceus var. mongholicus (Bunge) P.K. Hsiao (Astragalus mongholicus Bunge) has great utilization and development prospects. The main component of Astragalus mongholicus Bunge is Astragalus mongholicus polysaccharides (mAPS), of which the monosaccharide is composed of Mannose, Ribose, Arabinose, Glucose, Galactose, Xylose, Glucuronic acid and Galacturonic acid. The effects and mechanisms of mAPS against T2DM have not been fully clarified. In this study, we created a T2DM rat model via low-dose streptozotocin (STZ) injection after feeding with High-sugar and high-fat diet (HSHFD). The regulation of mAPS on glucose metabolism, hepatic inflammation, and gut microbiota was studied.

2. Materials and methods

2.1. Reagents and Reagents

Prof. Minhui Li identified and authenticated the plant roots of Astragalus mongholicus Bunge. A voucher specimen of Astragalus mongholicus Bunge (herbarium voucher number Mongol Huangqi-2018-0830) was stored in the Department of Pharmaceutical Engineering, College of Life Sciences, Inner Mongolia Agricultural University. Astragalus mongholicus polysaccharides (mAPS) were extracted and characterized in our earlier research.¹⁷ The regular diet (RD) and the high-sugar and high-fat diet (HSHFD) (chow diet 88 %, cholesterol 2 %, and lard 10 %) were purchased from Xiaoshu Youtai (Beijing, China), Metformin (MET) was obtained from Meilun Biology (Dalian, China). Streptozotocin (STZ) (S0130) was obtained from the Sigma (New Mexico, USA). Antibodies against Occludin (Lot: 10004180) and Zona Occludens 1 (ZO-1) (Lot: 10003932) were purchased from Proteintech (Wuhan, China). The antibodies against NF-κB (#8242), phospho-NF-κB (#3033), phospho-IκB (#2859), IκB (#4812), IRS (#2382), and phospho-AKT (#13038) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-TLR4 (WL00196), anti-GLUT4 (WL02425), anti-PI3K (WL03380), anti-AKT (WL0003b), anti-β-actin (WL01372) and anti-α-Tubulin (WL02296) were obtained from Shenyang Wan Lei Biotechnology Co., Ltd. (Shenyang, China). IR Dye 800 CW IgG (H + L) was purchased from LI-COR Biosciences (Nebraska, USA).

2.2. Preparation of mAPS

Polysaccharide were extracted from Astragalus mongholicus Bunge as described previously.^17,18 Briefly, the powder of Astragalus mongholicus Bunge (30 g) was extracted two times with distilled water (1:15, w/v) at 80 °C for 4h. The extracts were collected, filtered, concentrated, and then precipitated with 50 % ethanol at 4 °C for 24 h. The crude polysaccharide solution was dialyzed, concentrated, and lyophilized to obtain the polysaccharide extract (mAPS). Analysis of the monosaccharide composition in mAPS extract was carried out by using high-performance liquid chromatography (HPLC). There are 71.01 % carbohydrates, 3.28 % protein, 6.63 % Flavone, 1.45 % Ursolic acid, and 2.87 % Saponin in the mAPS.¹⁷ mAPS is composed of Mannose, Ribose, Arabinose, Glucose, Galactose, Xylose, Glucuronic acid, and Galacturonic acid (Table S1).

2.3. Animals and experimental Design

Male SD rats (6–8 weeks, 200 ± 20 g) were acquired from SPF Biotechnology Co., Ltd (Beijing, China). The animals were kept in an environment at 25 °C temperature, 50–60 % humidity, and under a light-dark cycle of 12 h, with free access to food and water. The animal procedures were approved by the Inner Mongolia Agricultural University's Experimental Animal Welfare Ethics Committee (No: NND2021092). Thirty SD rats were randomly divided into five groups (n = 6): Control (Ctrl) group, mAPS group, T2DM group, T2DM + mAPS group, and T2DM + MET group. The Ctrl and mAPS groups rats were given regular diets, while rats in other groups were fed with HSHFD. Four weeks later, rats fed with HSHFD were intraperitoneal injected with STZ (35 mg/kg). Fasting Blood glucose (FBG) levels consistently higher than 11.1 mmol/L can be considered a successful establishment of the T2DM model.¹⁹ Rats in the T2DM + mAPS and T2DM + MET groups received oral administration of 200 mg/kg of mAPS and 300 mg/kg of MET respectively, once a day for 4 weeks. All the rats that underwent 12 h fasting were anaesthetized via intraperitoneal injection with 1 % solution of pentobarbital sodium (0.17 mL/100g). After the samples of tissues, feces, and blood were collected, all the rats were euthanized via cervical dislocation. The weight of tissue samples of epididymal fat, colon, and liver were measured. 4 % paraformaldehyde was used to preserve a section of the liver and pancreas, while the rest tissues were promptly put in liquid nitrogen until further processing.

2.4. Biochemical analysis

The FBG levels in various group were monitored using a blood glucose meter (Bayer, Germany) at indicated time points. The following are the formulas used to calculate the HOMA-IR and insulin sensitivity index (ISI):²⁰

$$\text{HOMA}-\text{IR}=\frac{(\text{FBG})*(\text{FINS})}{22.5}$$

$$\text{ISI}=\frac{1}{(\text{FBG})*(\text{FINS})}$$

The levels of Triglyceride (TG), Total cholesterol (TC), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Low-density lipoprotein (LDL) and High-density lipoprotein (HDL) in serum were measured by using an automatic biochemical analyzer (Shizuoka, Japan). The serum levels of Interleukin-1β (IL-1β), Fasting insulin (FINS), and Tumor necrosis factor-α (TNF-α) were measured by the corresponding ELISA kits (NeoBioscience, Shenzhen, China).

2.5. Histoligical analysis

Following fixation in 4 % paraformaldehyde for over 24 h, the liver and pancreas tissues undergo gradually dehydration in varied concentrations of ethanol. Subsequently, the tissues were embedded with paraffin. The liver and pancreas tissues were divided into 5 μm slices, which were then stained with hematoxylin-eosin (HE). The criteria used for scoring liver and pancreas injury were followed as described previously.²¹

2.6. Real-time PCR analysis

The total RNA was extracted by using Trizol method, and the quality of RNA was evaluated with the NanoDrop2000 Spectrophotometer. The extracted RNA (1 μg) was then reversely transcribed to cDNA. The Real Time-PCR assay was performed by using the appropriate primers as shown in Table 1. The relative expression of specific genes was calculated by using the 2^−ΔΔCT method.

Table 1.

The primers used for Real-time PCR.

Gene name	Forward	Reverse
AKT	5′-TCACCTCTGAGACCGACACC	5′-GGCCTCCGTTCACTGTCCA
PI3K	5′-TGCTACAGGACTTCACGCAG	5′-GACTGTAGGATCGGCACGTT
GLUT4	5′-CTACGCCACCATAGGAGCTG	5′-GCCCAGCTCGCTCTACTAAG
IL-1β	5′-TGCTGTCTGACCCATGTGAG	5′-GTCGTTGCTTGTCTCTCCTTG
TNF-α	5′-ATCGGTCCCAACAAGGAGGA	5′-TCCGCTTGGTGGTTTGCTAC
NLRP3	5′-GTGGAGATCCTAGGTTTCTCTG	5′-CAGGATCTCATTCTCTTGGATC
TLR4	5′-TGAGGACTGGGTGAGAAATGAGC	5′-CTGCCATGTTTTGAGCAATCTCAT
IL-6	5′-CCACCAGGAACGAAAGTCAAC	5′-TTGCGGAGAGAAACTTCATAGCT
Actin	5′-CGCGAGTACAACCTTCTTGC	5′-ATACCCACCATCACACCCTGG

2.7. Western blotting

The liver tissue lysates were prepared by homogenization in Radio immunoprecipitation assay (RIPA) Lysis buffer (Beyotime, Biotech, China), supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified by using bicinchoninic acid (BCA) kits (Sangon Biotech, Shanghai, China). Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was used for protein separation before transfer to polyvinylidene fluoride (PVDF) membranes in a semi-dry transfer unit (Bio-Rad, USA). Following a 4 h blockage with non-fat milk powder at a concentration of 5 %, the membranes were incubated with primary antibodies (dilution ratio: 1:1000) at 4 °C overnight. After three times wash with Tris-buffered saline + Tween 20 (TBST), The PVDF membranes were then incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW, dilution ratio: 1:10,000) for 2 h. The image of proteins was captured and analyzed by using the Odyssey infrared imaging system (Li-COR, Nebraska, USA).

2.8. 16S rDNA gene sequencing

DNA in fecal samples from various group was obtained by utilizing the E.Z.N.A.®Stool DNA Kit. The V3–V4 variable region of 16S rDNA was amplified by utilizing primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′). The amplification process began with pre-denaturation at 98 °C for 30 s, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 54 °C for 30 s, extension at 72 °C for 45 s, and final extension at 72 °C for 10 min. After quantification, equivalent quantities of purified polymerase chain reaction (PCR) products were subjected to paired-end sequencing on the Illumina NovaSeq platform (Illumina, San Diego, CA, USA).

2.9. Bioinformatic analysis

The mentioned sequencing was carried out with the guidelines from the Lc-Bio Technologies (Hangzhou, China). Samples were assigned paired end reads based on their distinct barcodes and then truncated by removing the barcode and primer sequences. Fast length adjustment of short reads (FLASH) was used to merge the paired end reads. Quantitative insights into microbial ecology 2 (QIIME2) was applied to analyze α-diversity and β-diversity, and R (v3.5.2) software was used to create graphics. To compare the diversity of microbiota in the 18 fecal samples, the linear discriminant analysis (LDA) based LEfSe method was employed.

2.10. Statistical analysis

The R language was used to analyze and visualize the 16S rDNA sequencing results. GraphPad Prism 8.4 was used to perform analysis and graphing of data. SPSS17.0 software was adopted to perform statistical analysis. Differences were considered statistically significant when p < 0.05.

3. Results

3.1. The hypoglycemic effects of mAPS in T2DM rats

The body weight and FBG levels in all the groups of rats were measured during the entire experimental period. As shown in Fig. 1A, there is an increasing trend of body weight in all the groups of rats during the 30 days of feeding. Nevertheless, the rats in the T2DM group suffered mild weight loss in response to the STZ injection, and the body weights gradually increased after treatment with mAPS or MET. Furthermore, persistent high FBG levels were found in the rats challenged with STZ, while the levels of FBG notably decreased after mAPS administration (p < 0.05, Fig. 1B). Significant changes in the liver and fat index (Fig. 1C–D) were observed in the MET group as compared to the T2DM group (p < 0.05). Following 4 weeks of mAPS or MET intervention (Fig. 1B), the T2DM rats showed significantly lower FBG levels (p < 0.05).

Fig. 1.

mAPS administration improved IR in T2DM rats. (A) Body weight. (B) FBG levels after 4 weeks of mAPS treatment. (C) Liver index. (D) Fat index. (E) HOMA-IR. (F) ISI. Results are expressed as the mean ± SD. Data with different letters are significantly different (p < 0.05).

IR leads to a compensatory rise in insulin production and glucose uptake decrease. The HOMA-IR and ISI have been extensively employed for the purpose of guiding the management of T2DM.²² T2DM rats exhibited significantly higher HOMA-IR values than that of the Ctrl rats (p < 0.05). The rats that were administered with mAPS or MET showed significantly lower HOMA-IR values as compared to the T2DM rats (Fig. 1E, p < 0.05). The ISI values were restored to the control values with mAPS or MET treatment (Fig. 1F, p < 0.05). The result indicate that mAPS can improve T2DM-induced IR.

3.2. Effects of mAPS on lipid metabolic disorders and histopathological changes in T2DM rats

Fig. 2A–F illustrates that the T2DM group exhibited significantly elevated levels of TC, TG, LDL, ALT, and AST and a notable decrease in HDL level (p < 0.05). mAPS intervention dramatically reversed the trend in T2DM rats (p < 0.05), indicating that mAPS effectively alleviate lipid metabolism disorder in T2DM rats.

Fig. 2.

The effects of mAPS on serum metabolic parameters, liver and pancreas histopathology in T2DM rats. (A) TC, (B) TG, (C) ALT, (D) AST, (E) LDL, (F) HDL, (G) Representative HE staining of liver and pancreas (Original magnification: 200 × , scale bars = 50 μm), (H) Histological score of Liver, (I) Histological score of Pancreas. Results are expressed as the mean ± SD. Data with different letters are significantly different (p < 0.05).

Hepatic histopathological changes were assessed by HE staining. As shown in Fig. 2G, moderate to severe granular degeneration of the hepatocytes, lightly stained and loose cytoplasm (black arrow) was found in the T2DM group. In comparison, hepatocyte necrosis was evidently attenuated after treatment with mAPS. MET treatment also significantly reduced the injury scores of the liver (p < 0.05). The histological changes of the pancreas in different groups were observed subsequently. The pancreatic tissues in the T2DM group showed clear signs of degeneration (black arrow), blurred boundaries of islets, disordered arrangement of pancreatic cells, and infiltration of scattered inflammatory cells. In contrast, clear improvement of the morphology of the pancreatic cells and the structures of the pancreatic islets was found after mAPS or MET treatment.

3.3. Effects of mAPS on the gut barriers of T2DM rats

The tight-junction-associated protein Zona Occludens 1 (ZO-1) and Occludin levels were measured to evaluate gut barrier permeability. The expression levels of colon ZO-1 and Occludin mRNA in the T2DM group were significantly decreased (p < 0.05). However, the trend has been reversed after the supplementation of mAPS or MET. (Fig. 3A–B, p < 0.05). The Western blotting findings also indicated that mAPS treatment reversed the trend of decreased colon ZO-1 expression in the T2DM group (Fig. 3C, p < 0.05). No differences were found in the protein expression of Occludin between the T2DM and the T2DM + mAPS group (Fig. 3C). The notably increased serum LPS levels in the T2DM group were decreased after mAPS treatment (Fig. S2, p < 0.05), These results indicate that mAPS possess the ability to protect the integrity of the intestinal barrier.

Fig. 3.

The effects of mAPS on the expression of tight junction proteins in T2DM rats. (A, B) The mRNA levels of the ZO-1 and Occludin. (C) The protein expression colon of ZO-1 and Occludin. Results are expressed as the mean ± SD. Data with different letters are significantly different (p < 0.05).

3.4. Effects of mAPS on hepatic PI3K/Akt signaling pathways in T2DM rats

Glucose metabolism disorders in T2DM are always associated with deregulation of the PI3K/Akt signaling pathway and impaired GLUT4 translocation.²³ As shown in Fig. 4A–C, the mRNA levels of PI3K, AKT, and GLUT4 were markedly reduced in the T2DM rats, and increased after mAPS treatment (p < 0.05). Our further study showed that the hepatic protein levels of PI3K and phospho-AKT were significantly lower in the T2DM rats than that of the Ctrl rats (p < 0.05). The hepatic expression of phospho-PI3K was detected by the method of Immunohistochemistry, and not significant different was found among the studied groups (data not shown). There is also no significant change in the expression of IRS among the different groups (Fig. 4D). mAPS supplementation significantly enhanced the expression of phospho-AKT, PI3K, and GLUT4 in T2DM rats (Fig. 4D, p < 0.05). These findings suggest that the hypoglycemic properties of mAPS is associated with the regulation on the PI3K/AKT signaling pathway.

Fig. 4.

The effect of mAPS on the hepatic PI3K/AKT pathways in T2DM rats. (A–C) The mRNA expression of hepatic PI3K, AKT and GLUT4. (D) The protein expression of hepatic IRS, PI3K, GLUT4, p-AKT, and AKT. Results are expressed as the mean ± SD. Data with different letters are significantly different (p < 0.05).

3.5. Effects of mAPS on hepatic TLR4/NF-κB signaling pathways

Chronic inflammation is a major etiologic component of IR.²⁴ The TLR4/NF-κB signaling pathway has reportedly been related to metabolic inflammation and could serve as a promising target for treating T2DM.²⁵ We further studied the underlying mechanisms of the anti-inflammatory effects of mAPS against T2DM. As shown in Fig. 5, mAPS treatment significantly suppressed the production of serum IL-1β and TNF-α (Fig. 5A–B, p < 0.05). The transcription of IL-1β, TLR4, IL-6, TNF-α, and NLRP3 was elevated in the T2DM group and decreased after mAPS treatment (Fig. 5C, p < 0.05). It was found that the protein expression of TLR4, phospho-IκB, and phospho-NF-κB were all significantly elevated in the T2DM group. On the contrary, the tendency was reversed with mAPS supplementation (Fig. 5D, p < 0.05).

Fig. 5.

The effects of mAPS on the hepatic TLR4/NF-κB pathway in T2DM rats. (A–B) Serum IL-1β and TNF-α levels. (C) The mRNA expression of hepatic IL-1β, TNF-α, NLRP3, TLR4, and IL-6. (D) The protein expression of hepatic TLR4, p-NF-κB, NF-κB, p-IκB, and IκB. Results are expressed as the mean ± SD. Data with different letters are significantly different (p < 0.05).

3.6. Effects of mAPS on gut microbiota in T2DM rats

To evaluate whether the alleviating effect of mAPS against T2DM is related to the regulation of gut microbiota, 16S rDNA gene-sequencing technology was used to examine the microbial community diversity in fecal samples. The Venn diagram analysis indicated that the T2DM + mAPS rats showed a lower OTU abundance than that of the rats in the T2DM group (Fig. 6). No significant difference in the gut bacterial community's α-diversity and β-diversity were found between the T2DM and T2DM + mAPS rats.

Fig. 6.

Microbial diversity analysis in T2DM rats after mAPS intervention. (A) Venn diagrams. (B) α-diversity analysis of gut microbiota based on Shannon indexes. (C) β-diversity analysis of gut microbiota based on PCA.

To further examine the alterations in the structure of the gut microbiota, the proportions of bacteria at the phylum and class levels were studied. As shown in Fig. 7A, the composition of the microbial community structure mainly consisted of Firmicutes and Bacteroides, which accounted for 70 % and more of the relative abundance at the phylum level. The T2DM rats exhibited a greater proportion of F/B, while mAPS intervention reduced this ratio (Fig. 7D, p < 0.05). Notably, there is a notable reduction in the enrichment of Proteobacteria in the T2DM + mAPS rats. Clostridia and Bacteroidia comprise over 80 % of the relative abundance in class. Our data showed that 21 out of 30 classes were identified and changed significantly following mAPS treatment (Fig. 7B). As shown in Fig. 7C, the abundance of Clostridia in T2DM rats was significantly elevated, and this phenomenon was reversed after mAPS treatment (p < 0.05). In addition, the relative abundance of Bacteroidia in the T2DM rats was less abundant than that of the T2DM + mAPS rats.

Fig. 7.

mAPS-regulated gut microbiota composition in T2DM rats. (A) Composition of bacteria phylum levels. (B) Composition of bacteria class levels. (C) The relative abundance of Firmicutes, Bacteroides, Proteobacteria, Clostridia. (D) The ratio of Firmicutes/Bacteroides. Results are expressed as the mean ± SD. Data with different letters are significantly different (p < 0.05).

In addition, the least discriminant analysis (LDA) showed that the abundance of Ruminococcaceae, Proteobacteria, and Lachnospiraceae displayed a visible alteration among the different groups (Fig. 8). mAPS treatment substantially increased the abundance of the Ruminococcaceae, Bacteroides, and Lachnospiraceae. The abundance of Proteobacteria and Alistipes were significantly increased in the T2DM group, while the trend was reversed after mAPS treatment. These results indicated that mAPS supplementation affect the gut microbiota structure in T2DM rats.

Fig. 8.

LEfSa analysis of gut microbiota composition in T2DM rats after mAPS treatment. (A) LEfSa multi-level classification tree diagram. (B) LDA-discriminant histogram (LDA >4).

The relationship between the genus levels of gut microbiota and metabolic parameters was performed by using Spearman's correlation. As shown in Fig. 9, the abundance of Lachnoclostridium, Ruminococcaceae_UCG-014, and Prevotellaceae_NK3B31 were positively correlated with ISI, HDL, and the mRNA transcription levels of AKT, PI3K, GLUT4. In contrast, the abundance of Clostridium, Bilophila, and Parabacteroides was positively correlated with the liver index, TG, ALT, HOMA-IR, and transcription levels of IL-6 and IL-1β. these indicators were strongly correlated with IR, inflammation, and glucose metabolism disorders.²⁶

Fig. 9.

Correlation analysis between metabolic parameters and gut microbiota at the genus level. Note: *: p＜0.05，**: p＜0.01, ***: p＜0.001.

4. Discussion

Our previous results showed that mAPS exhibited protective effects against dexamethasone-induced metabolism disorder¹⁸ and HFD-induced NAFLD.¹⁷ This study aimed to further explore the hypoglycemic effects and underlying mechanisms of mAPS against T2DM. Our results indicated that mAPS could alleviate glucose metabolism disorder by modulating the PI3K/Akt, TLR4/NF-κB pathways, and gut microbiota. mAPS could be a promising medication for treating T2DM. In recent years, plant polysaccharides are widely used in clinical applications. Hu et al. study²⁷ found that the seaweed polysaccharides can improve the quality of life of patients with coronary heart disease and diabetes, and Li et al. study²⁸ found that astragalus polysaccharides are effective in the prevention of cardiotoxicity. Based on our findings, mAPS is expected to promote inter-clinical application after going on clinical trials. Moreover, mAPS could be applied to the development of nutraceuticals for regulating glycolipid metabolism. Our previous study found that mAPS at 50mg/kg, 100mg/kg, and 200mg/kg, could dose-dependently alleviate dexamethasone-induced disorder in glucose and lipid metabolism.¹⁸ Our further study demonstrated that administration of mAPS at a dose of 200mg/kg alleviates hepatic inflammation and reduces lipid accumulation in NAFLD rats.¹⁷ In this study, the dose of 200mg/kg was used to evaluate whether the same dose of mAPS has effect on insulin resistance.

The T2DM model of rats was characterized by high blood glucose and IR after challenged with HSHFD and STZ.²⁹ Our T2DM model rats also showed the same symptoms after fed with HSHFD for 4 weeks plus subsequent STZ injection. mAPS intervention decreased the levels of HOMA-IR and increased the ISI index (Fig. 1). We further found that mAPS effectively decreased serum lipid levels, mitigated hepatic degeneration and necrosis as shown in HE staining (Fig. 2). The finding is consistent with recent studies in which APS could restore glucose homeostasis and insulin resistance.^30,31

Numerous researches have shown that insulin receptor plays crucial roles in glucometabolism by binding to its transmembrane receptor to activate downstream PI3K/Akt signaling pathway.32, 33, 34 IR leads to the blockade of the PI3K/AKT signaling pathway, which further affects insulin-mediated physiological actions.³⁵ Modulation of the PI3K/AKT pathway become a promising approach for the management of T2DM.^36,37 It is well shown that insulin induced Akt activation can enhance GLUT4 transportation and promote the uptake of glucose.³⁸ In our study, the hepatic expression of GLUT4 decreased in the T2DM group and increased after mAPS intervention (Fig. 4). Our data is consistent with previous researches in which activation the PI3K/Akt pathway and increased GLUT4 expression were involved in the beneficial effects of several polysaccharides against T2DM.39, 40, 41 Our results demonstrated that mAPS improve glycolipid metabolism and IR in T2DM via activating of the PI3K/Akt pathway.

Evidence has shown that the damage of tight junction protein ZO-1 and Occludin is highly associated with systemic inflammation.⁴² Our study showed that the levels of serum LPS, TNF-α, and IL-1β in the T2DM rats were significantly increased, indicating there is systemic inflammation along with the development of T2DM. The result is in line with that of Chen et al.⁴³ The treatment of mAPS leads to reduced production of inflammatory mediators and increased expression of ZO-1 and Occludin (Fig. 3). These findings indicate that mAPS supplementation improve the integrity of intestinal barrier. LPS can be specifically recognized by receptor TLR4, leading to the activation of the NF-κB pathway and promoting the expression of multiple inflammatory mediators.^44,45 In this research, it was discovered that the T2DM rats exhibited elevated levels of TLR4, phospho-IκB, phospho-NF-κB, TNF-a, IL-6, and IL-1β, However, mAPS treatment attenuated the inflammatory response (Fig. 5). Our results agree with the experimental results from Liu et al.⁴⁶ And thus, we consider that the anti-inflammatory effects of mAPS are associated with down-regulation of the TLR4/NF-κB signaling pathway.

Gut microbiota dysbiosis is always accompanied with the development of T2DM.^47,48 Zhang et al.⁴⁹ found that the abundance of Firmicutes increased, while the abundance of Bacteroidetes decreased in patients with Diabetic Nephropathy. Our data also showed that the administration of mAPS modulated the ratio of Firmicutes/Bacteroidetes (Fig. 7). Furthermore, significant changes in the abundance of Proteobacteria, Ruminococcaceae_UCG-005, Muribaculaceae, and Lachnospiraceae-NK4A136 have been found in T2DM rats after mAPS supplementation (Fig. 8). Proteobacteria has been reported to be linked with gut microbiota disorder or instability in T2DM.^50,51 In our study, the increase in the abundance of Proteobacteria in the T2DM group was significantly reduced with mAPS intervention (Fig. 7). Meanwhile, mAPS treatment dramatically increased the abundance of Muribaculaceae, which is negatively associated with impaired glucose tolerance and lipid accumulation.⁵² In addition, we found that the abundance of Lachnospiraceae-NK4A136 in the T2DM group was significantly lower than that of the T2DM + mAPS group. This result agreed with the study by Zhu et al.⁵³ We further evaluated the association among PI3K/AKT, TLR4/NF-κB signaling pathway, and gut microbiota by the method of Spearman's correlation analysis. The analysis showed that Ruminococcaceae_UCG-005 and Lachnospiraceae-NK4A136 are positively correlated with ISI, HDL, AKT, and GLUT4, while negatively correlated with HOMA-IR, TG, ALT, IL-6, IL-1β, and TLR4. Lachnospiraceae-NK4A136 was reported to be associated with reduced inflammation, and improved insulin resistance.⁵⁴ A previous study by Zhao et al.⁵⁵ also suggested that Lachnospiraceae-NK4A136 was negatively correlated with the expression of TLR4, which is consistent with our study. To sum up, we consider there is a possible complicated interdependence among gut microbiota and PI3K/AKT, TLR4/NF-κB. Supplementation with mAPS affects this whole system.

5. Conclusions

In this study, we demonstrated that mAPS could alleviate the symptoms in rats of T2DM induced by combined challenge of HSHFD and STZ. The effects are association of activating the PI3K/AKT pathway, inhibition of the TLR4/NF-κB pathway, and modulation the gut microbiota. Nonetheless, the main components that contribute to the beneficial effects of mAPS against T2DM remain unclear. In addition, the interactions between mAPS and microbiota and the effects of mAPS on microbiota metabolites are in need of further exploration. Considering the difference among animal species, further clinical studies are required to verify the mechanisms of mAPS against T2DM in order to establish its clinical utility.

Author contributions

Haisheng Yuan: conceptualization, methodology, investigation, data curation, and writing-original draft. Guoquan Xu&Yan Yan: investigation, data curation, and writing-review and editing. Jingran Liu & Shimin Zhao: software, validation, formal analysis. Fujuan Cai &Xiuling Yu: visualization, Writing-review & editing. Yuzhen Wang & Minhui Li: supervision and Writing-review & editing. All authors have read and agreed to the revised version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No. 82160776), Natural Science Foundation of Inner Mongolia (2023LHMS08074), Inner Mongolia Innovation Team Development Program (NMGIRT2219), Science and technology programs of Inner Mongolia (2023YFDZ0029) and Basic scientific research business of colleges and universities directly under the Inner Mongolia Autonomous Region (BR231404).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

figs1.

figs2.