Stachyose improves the anti-diabetic effects of berberine by regulating intestinal microbiota and SCFAs in spontaneous type 2 diabetic KKAy mice

Background: Berberine (BBR) has the bene�cial effects of anti-inammation, anti-bacteria, and anti-diabetes. The clinical application of BBR has been hindered by its poor gastrointestinal absorption. Stachyose (Sta), a prebiotic agent, improves the composition of gut microbiota and bene�ts for diabetes. We therefore investigated whether Sta improves the anti-diabetic actions of BBR using type 2 diabetic KKAy mice. Methods: KKAy mice were randomly divided into four groups: Con, Sta (200 mg/kg), BBR (100 mg/kg), and BBR+Sta (BBR: 100 mg/kg, Sta: 200 mg/kg). Mice were administered intragastrically once daily for 8 weeks. The mice weight, glycemia, glycated hemoglobin, and levels of insulin, glucagon, and in�ammatory cytokines were monitored. Oral glucose tolerance tests and insulin tolerance tests were performed; protein and gene expression levels were detected by Western blot and qRT-PCR, respectively; gut microbiota and short-chain fatty acids (SCFAs) in feces were analyzed. Results: The combination of BBR and Sta is more effective than BBR alone in blood glucose control, improvement of insulin resistance and islet functions, in�ammatory mediators decrease, and maintenance of intestinal barrier integrity. Gut microbiota analysis demonstrates that both BBR and combination treatments enhance the abundance of Bacteroidaceae and Akkermansiaceae and decrease Lachnospiraceae levels, whereas Akkermansiaceae elevation due to the administration of BBR with Sta is more signi�cant than BBR alone. Interestingly, the proportion of Lactobacillaceae increases with combination treatment, but is diminished by BBR treatment. Additionally, BBR with Sta signi�cantly reduces the concentrations of fecal SCFAs compared to BBR. Conclusions: The combination of BBR and Sta imparts better effects on the maintenance of glycemia and intestinal homeostasis than BBR alone by modulating gut microbiota and SCFAs, thereby providing a novel approach for the treatment of type 2 diabetes mellitus.


Background
Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by obesity, insulin resistance, chronic hyperglycemia, low-grade in ammation, alteration of gut microbiota, and islet β cell dysfunction [1].T2DM, which accounts for 90% of diabetes cases, is a major economic burden and life pressure on people.In addition, the incidence of diabetes continues to increase each year.Hence, it is essential to identify new preventive approaches to inhibit the progression of T2DM and control the growing epidemic.
Traditional Chinese medicine has been used in clinic for over 2,000 years.The Qianjin huanglian pill, which is composed of Coptis chinensis powder and fresh Rehmanniae radix, is a classic ancient prescription for treating diabetes due to its effects of nourishing Yin and clearing heat.Berberine (BBR), one of the main components of R. coptidis, is an isoquinoline alkaloid originally isolated from C. chinensis [2].Several studies have shown that BBR has many advantageous biological roles in preclinical and clinical research, such as anti-in ammation, anti-insulin resistance, anti-hyperglycemia, anti-hyperlipidemia, anti-bacteria, and anti-diabetes [3,4].BBR also ameliorates liver, cardiovascular, and renal disease that are associated with diabetes [3].This evidence suggests that BBR is a potential drug for the treatment of T2DM.However, BBR may result in gastrointestinal reactions (including diarrhea and constipation) in some patients due to its poor absorption, thereby limiting its long-term and wide application in T2DM management [3,5] .
A plethora of studies has revealed that prebiotics not only reduce the in ammatory response and oxidative stress mainly by enhancing the growth of speci c bene cial bacteria found in the gut, but also improve intestinal tight junction integrity and decrease intestinal permeability by increasing the expression of adhesion proteins within the intestinal epithelium, consequently preventing the occurrence and development of T2DM [6,7].Furthermore, prebiotics have the ability to promote the absorption of antioxidants [6].Reports have also shown that BBR exhibits anti-diabetic actions by regulating gut microbiota composition and SCFAs [8,9].On the basis of these ndings, we hypothesized that usage of a prebiotic during BBR treatment may optimize glucose metabolism and better protect against diabetes.
Stachyose (Sta) is a functional oligosaccharide extracted from fresh R. radix.As a classic prebiotic, Sta has been proven to promote the growth of bene cial intestinal bacteria, inhibit pathogenic bacteria, and improve diabetes [10,11].Moreover, Sta enhances the absorption of soybean genistein and tea polyphenols in mice [12,13].Therefore, we hypothesized that Sta improves the anti-diabetic actions of BBR by regulating intestinal microbiota.This study aimed to investigate the effects and mechanisms of BBR combined with Sta on glucose metabolism, in ammation modulation, intestinal integrity, fecal SCFAs, and gut microbiota in diabetic KKAy mice.

Chemical compounds
Sta (purity>80%) was provided by the laboratory of Professor Dequan Yu, Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union Medical College (Beijing, China).BBR (purity>98%) was obtained from the Northeast General Pharmaceutical Factory (Shenyang, China).

Animals and experimental design
Eight-week-old female KKAy mice were purchased from Beijing Huafukang Bio-Technology Co., Ltd., and maintained at Experimental Animal Center of the Institute of Materia Medica under speci c pathogen-free conditions.Animals were kept at 23 ± 2℃ in a 12-h light/dark cycle with free access to food and water.According to the levels of blood glucose, triglyceride, total cholesterol, body weight, and percentage of glycemia reduction at 40 min after insulin injection, KKAy mice were randomly divided into four groups (n=12): control group (Con), Sta-treated group (Sta, 200 mg/kg), BBR-treated group (BBR, 100 mg/kg), and BBR with Sta-treated group (BBR+Sta; Sta: 200 mg/kg, BBR: 100 mg/kg).All mice were treated intragastrically with compounds or an equivalent volume of water once daily for 8 weeks.
Fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) measurements FBG levels were measured using the glucose-oxidase method (Biosino Bio-Technology & Science Inc., Beijing, China).After 35 days of treatment, HbA1c levels were assessed using commercial kits (Homa Biological, Beijing, China) Oral glucose tolerance test (OGTT), insulin tolerance test (ITT), and glucose-stimulated insulin secretion test For OGTT, mice were fasted for 4 h and given D-glucose (2 g/kg) intragastrically, and blood glucose was measured at 0, 15, 30, 60, and 120 min after glucose administration.For ITT, mice were injected subcutaneously with insulin (0.4 U/kg) after 4 h of fasting, and blood glucose was measured at 0, 40, and 90 min after insulin injection.For glucose-stimulated insulin secretion test, mice were treated with Dglucose (2 g/kg) after 4 h of fasting, and insulin levels in plasma were monitored at 0 and 15 min after glucose administration.

Immuno uorescence assay
All mice were sacri ced through cervical dislocation and the pancreas were xed in 10% formalin, embedded in para n, and dissected to prepare 5-μm-thick slides, then stained with antibodies against insulin and glucagon as previously described [14].The mean uorescence intensities of insulin and glucagon, which account for islet areas were quanti ed using Image J software.
The concentrations of glucagon and insulin were determined by ELISA kits according to the manufacturer's instructions (ALPCO, USA).
Quantitative real-time PCR (qRT-PCR) qRT-PCR was conducted as previously described [15].Brie y, RNA was isolated from intestinal tissues using TRizol reagent and reverse transcribed with TransScript® rst-strand cDNA Synthesis SuperMix (TransGen Biotec, Beijing, China) based on the manufacturer's protocols.qRT-PCR was conducted using TransStart® Tip Green qPCR SuperMix (TransGen Biotec, Beijing, China) on 7900 Real-Time PCR System (Applied Biosystems, USA).Gene expression levels were normalized to those of β-actin.The primer sequences used in this study are shown in Table S1.

Gut microbiota pro ling
The intestinal ora in feces was assayed as previously described [16].Fecal samples were collected and snap-frozen in liquid nitrogen, followed by storage at -80℃.Genomic DNA was isolated with DNA isolation kit.The V3-V4 region of 16S rRNA genes were ampli ed and puri ed.Subsequently, the abundance and diversity of gut microbiota were analyzed using Illumina MiSeq sequencing (Major Bio-Pharm Technology, Shanghai, China) according to the standard protocol.The effective reads from all samples were grouped into operational taxonomic units (OTUs) on the basis of 97% sequence similarity.α-Diversity was estimated by the level of OTUs, and Shannon and Chao indices.β-Diversity was assessed by computing for unweighted UniFrac and visualized by principal coordinate analysis (PCoA).The sequence data were processed and analyzed on the free online Majorbio I-Sanger Cloud Platform (www.isanger.com).

Short-chain fatty acids (SCFAs) analysis
SCFAs in fecal samples were detected on the basis of previous report [17].Brie y, frozen faeces were crushed with methanol containing internal standards, subjected to ultrasonication for 30 min, centrifuged for 15 min, and extracted.Subsequently, the SCFAs in each sample were assayed by gas chromatography coupled to a mass spectrometer detector (GC-MS) (Agilent Technologies Inc. CA, USA) and quanti ed using Masshunter quantitative software.
Correlation analysis of SCFAs and gut microbiota were performed on the platform of Majorbio I-Sanger Cloud (www.i-sanger.com).R and P values were obtained using Spearman's rank correlation.

Statistical analysis
The data are presented as the mean ± SEM.Statistical analysis was performed using one-way ANOVA and unpaired two tailed Student's t-test, and plotted using GraphPad Prism 7.0.Differences with p < 0.05 were considered statistically signi cant.

Effects of berberine with stachyose on glucose homeostasis
In this study, we found that the levels of blood glucose and HbA1c in mice treated with either BBR or BBR+Sta were signi cantly decreased compared to control mice (Fig. 1a, b).Further, HbA1c levels in combination-treated mice obviously lowered compared to those of BBR-treated mice (Fig. 1b), indicating that BBR combined with Sta is more effective in glycemic control than BBR alone.
In comparison to the Con mice, supplementation with BBR or BBR with Sta improved glucose tolerance and insulin sensitivity, as evaluated by diminished AUCs for blood glucose during OGTT and ITT (Fig. 1c,  d).We further detected the index of HOMA-IR to estimate insulin resistance levels.The HOMA-IR indices in Sta, BBR, and BBR with Sta-treated mice were decreased by 21.9% (p = 0.0880), 61.2% (p < 0.001), and 79.3% (p < 0.001), respectively, compared to the Con mice (Fig. 1e), which implied that BBR with Sta is better in improving insulin sensitivity of KKAy mice than BBR alone.
In addition, treatment with BBR or BBR with Sta resulted in lower weight gain, food consumption, and water intake (Fig. 1f, Fig. S1).Therefore, the effects of BBR and BBR with Sta on body weight may be in correlation with decreased food intake.We noticed that the food and water intake of mice had no distinction between BBR and combination groups, but the weight of mice administrated of BBR with Sta was higher than that of BBR treatment at 8 th , 26 th , and 35 th days.Thus, Sta may attenuate the gastrointestinal side effects of BBR, bringing about mild weight increase.

Effects of berberine with stachyose on islet functions
As shown in Fig. 2a, compared to the Con group, fasting plasma insulin levels in the BBR and BBR with Sta groups were reduced by 27.8% (p < 0.05) and 45.7% (p < 0.01), respectively.Besides, BBR with Sta treatment promoted insulin release and increased proportion of insulin elevation from 17.5% in Con mice to 118.2% (p < 0.05), while BBR treatment elevated to 76.0% (p < 0.05) (Fig. 2a-c).And the concentration of plasma glucagon was only reduced by the combined treatment in comparison to Con or BBR supplementation (Fig. 2d).
Compared to the Con group, glucagon contents in islets were decreased in the BBR combined with Sta group and had no obvious alteration in the BBR group, and insulin levels in the islets of all treatment groups were declined (Fig. 2e, f).Additionally, the mean uorescence intensity ratio of insulin to glucagon was markedly increased in the combination group (Fig. 2f).Taken together, these results suggest that BBR combined with Sta is more effective than BBR alone in maintaining insulin-glucagon homeostasis and ameliorating islet functions.

Effects of berberine with stachyose on in ammation
Compared with the Con mice, treatment with BBR and BBR with Sta diminished cytokine levels of IL-1β, TNF-a, IL-6, CRP in plasma, Sta reduced IL-1β levels and increased IL-10 levels, while the production of MCP-1 was only decreased by BBR with Sta treatment (Fig. 3a).Similarly, the gene levels of proin ammatory cytokines-IL-1β, TNF-a, MCP-1, and IL-6-were attenuated by BBR and BBR with Sta, whereas anti-in ammatory cytokine IL-10 levels in intestine was elevated (Fig. 3b).And the actions induced by the combination treatment were more pronounced (Fig. 3b).Given that the production of proin ammatory cytokines is related to the activation of the TLR4 and MAPK signaling pathways [18], we next examined whether these pathways in intestinal tissues were impacted by BBR with Sta treatment.As shown in Fig. 3b-d, compared to the Con group, the protein and mRNA levels of TLR4 as well as the phosphorylation of ERK and p38MAPK were all suppressed in the BBR and BBR with Sta groups, without obvious change of JNK phosphorylation.

Effects of berberine with stachyose on intestinal integrity
Given that in ammation contributes to intestinal dysbiosis and damages gut permeability, we explored the intestinal integrity of KKAy mice.Compared to the Con group, the protein and gene expression levels of ZO-1 and occludin, which are tight junction components, were upregulated in the BBR and BBR with Sta groups, and Sta remarkably elevated ZO-1 and occludin gene levels (Fig. 4a-c).Moreover, relative to the Con mice, the mRNA expression of Reg3g (regenerating islet-derived 3 gamma), an antimicrobial protein was increased by 1.4-od (p > 0.05) and 1.7-fold (p < 0.01) in mice treated with BBR and BBR with Sta, respectively (Fig. 4d).These data indicate that BBR combined with Sta is more effective than BBR alone in maintaining gut barrier integrity and intestinal balance.

Effects of berberine with stachyose on gut microbiota
We next examined the roles of BBR with Sta on intestinal microbiota composition by performing Illuminasequencing based analysis of bacterial 16S rRNA in fecal samples.After removing unquali ed sequences, a total of 35,299 valid sequences were generated and clustered into 316 OTUs according to the minimum sample-sequence number (Table S2).Compared to the Con group, the OTU numbers were reduced in the BBR and BBR with Sta groups (Fig. 5a).And the OTU numbers were remarkably less in the combined group than those of the BBR group (Fig. 5a).The Shannon and Chao indices re ect the diversity and richness of gut microbiota, respectively.Compared to the Con group, BBR and BBR with Sta diminished the indices of Shannon and Chao, whereas there were notable differences in the Chao index between the BBR and BBR with Sta groups (p < 0.01) (Fig. 5b, c).
Unweighted Unifrac PCoA based on OTU levels revealed a distinct clustering of microbiota composition in each group (Fig. 5d).Multivariate analysis of variance of PCoA matrix scores revealed that the microbiota community of mice in BBR and combination groups is clearly differed from that of the Con mice.The gut microbiota of the Sta, BBR, and BBR with Sta groups could also be discriminated (Fig. 5d).Additionally, the bacterial community of Sta-administrated mice was closer to that of the Con mice, which coincides with the mild actions exerted by Sta.
Taxonomic pro ling at the phylum level revealed that BBR and BBR with Sta treatments elevated the abundances of Verrucomicrobia and reduced that of Deferribacteres and Epsilobacteraeota compared with the Con mice (Fig. 5e).Notably, the increase in abundance of Verrucomicrobia produced by BBR with Sta was more signi cant than BBR alone.At the same time, Sta decreased the levels of Deferribacteres and Proteobacteria (Fig. 5e).At the family level, the abundances of Bacteroidaceae and Akkermansiaceae were notably enhanced, and Lachnospiraceae and Desulfovibrionaceae were decreased in the BBR and BBR with Sta groups compared to the Con group (Fig. 5f).In addition, the proportion of Akkermansiaceae in combination-treated mice was higher than that of BBR-treated mice.Strangely, the proportion of Lactobacillaceae was diminished by BBR but increased by the combination of BBR and Sta (Fig. 5f).The levels of Desulfovibrionaceae were also diminished by Sta treatment.Similar results were also observed at the genus level.Combination treatment declined Desulfovibrio abundance, and increased the abundance of Lactobacillus and Akkermansia compared with BBR treatment (Fig. 5g).Collectively, these ndings implied that Sta augments the anti-diabetic effects of BBR and attenuates its adverse reaction by regulating the composition of gut microbiota.

Effects of berberine with stachyose on fecal SCFAs
SCFAs, including acetic acid, propionic acid, butyric acid, isobutyric acid, pentanoic acid, isopentanoic acid, hexanoic acid, isohexanoic acid, and total SCFAs, were assessed in this study.Compared to the Con group, acetic and propionic acids were elevated, whereas hexanoic, isohexanoic, and isopentanoic acids decreased in the Sta group (Fig. 6a).Simultaneously, BBR reduced the contents of total SCFAs, butyric, pentanoic, isopentanoic, hexanoic, and isohexanoic acids in feces (Fig. 6a).In addition, the eight SCFAs and total SCFAs were all obviously downregulated in the BBR with Sta group compared with the Con or BBR group (Fig. 6a).Overall, BBR combined with Sta signi cantly diminished fecal SCFAs concentrations in KKAy mice.Subsequently, we analyzed the relationship of fecal SCFAs and intestinal bacteria at the family level (Fig. S2, Table S3).The results displayed that Bacteroidaceae and Akkermansiaceae were negatively correlated with acetic acid, propionic acid, butyric acid, isobutyric acid, pentanoic acid, and total SCFAs, while Lachnospiraceae was positively related with these SCFAs (Fig. 6b, Table S3).And a signi cant positive correlation between Desulfovibrionaceae and pentanoic acid was observed (Fig. 6b, Table S3).These results were consistent with the above results of microbiota and SCFAs, which suggest that BBR with Sta improves blood glucose, in ammation, and gut integrity is linked to the alteration of intestinal microbiota and SCFAs.

Discussion
Diabetes has become a major public health concern because it causes various health problems and its global prevalence has increased in recent decades [19].BBR has various pharmacological properties and multispectral therapeutic applications, including diabetes, hyperlipidemia, and metabolic syndrome [3].Nevertheless, the use of BBR is limited due to its poor absorption and gastrointestinal adverse effects.Sta, a functional prebiotic, has been shown to improve antioxidants absorption and intestinal microbiota composition, leading to the ameliorated in ammation and diabetes [10,11,20].Here, we observed that combination of BBR and Sta is more effective than BBR alone in modulating glucose metabolism, gut in ammation, and intestinal barrier integrity by altering gut microbiota and SCFAs in the stool of KKAy mice.
T2DM is accompanied with increased levels of blood glucose and HbA1c, as well as islet dysfunction.
HbA1c is an index of the control of diabetes and re ects the average blood glucose levels over the past two-three months [21].Glucose homeostasis is maintained by insulin and glucagon, whereas disrupted pancreatic islet functions and insulin resistance in T2D leads to glucose intolerance [21].In this study, our ndings showed that the combination treatment of BBR and Sta initiated better bene cial actions on glycemia control and islets functions improvement compared to BBR alone.Furthermore, the weight of mice received the combination of BBR and Sta was higher than that of BBR-treated mice at conditions of equal food consumption.Therefore, BBR combined with Sta produces better effects on modulating glycemia homeostasis than BBR alone, which is correlated with relieved gastrointestinal side effects of BBR and modi ed islet functions.
It is widely accepted that T2DM is typical of chronic low-grade in ammation of metabolic tissues, such as adipose, liver, intestine, and islets [22].And various in ammation-related molecules are associated with the development of T2DM, like TLR4, MAPK, and NF-κB [23].Clinical studies have shown that when in ammation in T2DM patients is suppressed by a high dose of aspirin or salsalate, the glycemic control of the patients improves, along with concomitant inhibition of NF-κB activity [23].Our ndings revealed that BBR combined with Sta imparts stronger effects on abating the expressions of proin ammatory mediators in plasma as well as in the intestines than those of BBR alone.In addition, BBR with Sta treatment downregulated the activation of TLR4 signaling pathway and the phosphorylation of ERK and p38MAPK.Overall, treatment using the combination of BBR and Sta alleviates in ammation of KKAy mice at least partially through the regulation of TLR4, ERK, and p38MAPK pathways.
Intestinal dysbiosis results in increased intestinal permeability and susceptibility to microbial antigens, which ultimately contribute to the occurrence and development of in ammation and diabetes [6].ZO-1 and occludin acting as tight junction proteins play protective roles in intestinal permeability [24].T2DM is usually accompanied with increased intestinal permeability characterized by reduction in tight junction proteins [25].And accumulation of these molecules is associated with increased protection at the intestinal barrier level.Herein, we found that treatment with the combination of BBR and Sta produced higher expressions of occludin and ZO-1 than BBR supplementation.Sta treatment also augmented these molecules expressions.What's more, prebiotics decrease intestinal permeability and maintain gut barrier integrity by modulating gut microbiota composition, ultimately inhibiting T2DM progression [7].Therefore, our results suggest that Sta as a prebiotic enhances the positive effects of BBR in maintaining gut barrier integrity of KKAy mice, which may be attributed to speci c alterations in the gut microbiota.
The major alterations in the intestinal microbiota that are linked to T2DM include a signi cantly lowered prevalence of Bacteroidetes and Verrucomicrobia and an enrichment of Firmicutes, Proteobacteria, and Deferribacteres [26].Although the ratio of Bacteroides to Firmicutes was not altered in the study, Bacteroidaceae, belonging to Bacteroides, was obviously increased by BBR and BBR with Sta.
Importantly, combination treatment enhanced Verrucomicrobia abundance compared with BBR alone.In line with these results, in comparison to BBR, the levels of Akkermansia and Akkermansiaceae, belonging to Verrucomicrobia, were also markedly increased by BBR with Sta.Akkermansia has bene cial effects on glucose metabolism and gut permeability, which is negatively associated with T2DM and has been tested as a probiotic in preclinical trials [27].Plovier et al. [28] found that pasteurized Akkermansia muciniphila versus the live bacteria has an enhanced capacity to reduce fat mass development, insulin resistance and dyslipidemia in mice.Our previous study also manifested that BBR with Sta increased the abundances of phylum Verrucomicrobia and species Akkermansia muciniphila in the feces of db/db mice [4].In addition, Verrucomicrobia is highly abundant in healthy subjects [29].Thus, the increased proportion of the phylum Verrucomicrobia and genus Akkermansia induced by BBR with Sta contributes to the amelioration of glucose metabolism and intestinal integrity.
Lactobacillus genus acting as probiotic is bene cial to healthy intestinal microbiota, creates a favorable intestinal environment, and alleviates T2DM [30].We observed that BBR treatment reduced the levels of Lactobacillaceae family and Lactobacillus genus, whereas BBR combined with Sta reversed the reduction to a higher percentage than those seen in control mice.The abundances of Desulfovibrio genus were elevated in obesity and T2DM [31].Desulfovibrio which is considered an opportunistic pathogen produces endotoxins and have the capacity to reduce sulfate to H 2 S, thereby damaging the intestinal barrier [31].We here found that both BBR and combined treatments reversed these elevations, and combined treatment diminished the Desulfovibrio genus level compared with BBR alone.Furthermore, it has been demonstrated that Akkermansia and Lactobacillus protect against in ammation [27].Taken together, Sta enhances the e cacy of BBR against diabetes through altering gut bacterial composition and maintaining intestinal homeostasis.
SCFAs are closely associated with obesity and diabetes, which are products of gut microbiota mediated fermentation of resistant starch or dietary ber [17].Firmicutes and Bacteroidetes are the predominant intestinal bacteria that produce SCFAs [32].Bacteroidetes mainly produces acetate and propionate, whereas butyrate is mostly generated by Firmicutes [37].In this study, we found that the abundance of Firmicutes and Bacteroidetes did not signi cantly differ among the study groups, and thus the production of SCFAs in the mouse groups is most likely the same.Interestingly, our results showed that fecal SCFA reduction in mice that received the combination treatment was remarkably less than that of mice treated with BBR alone, which is contradictory too the results of recent studies that prebiotics and berberine elevated SCFAs concentrations to alleviate obesity and diabetes [33,34].However, there are animal and human studies suggested that an increased concentrations of SCFAs in feces was associated with higher body weight and fat gain and insulin resistance, which may be due to increased SCFAs production and decreased SCFAs absorption [35].Therefore, decreased fecal SCFAs in KKAy mice generated by BBR with Sta is most probably bene cial in protecting against the development of T2DM.
SCFAs exert a number of bene cial effects to improve gut health, insulin resistance, and diabetes , as well as enhance intestinal barrier function by regulating the expression of tight junction proteins and mucous [36].SCFAs, particularly propionate and butyrate also inhibit the maturation of monocytes and macrophages, altering their abilities to capture antigens and reducing their ability to produce proin ammatory cytokines such as IL-1β, IL-6, and TNF-α [37,38].Nonetheless, most SCFAs are absorbed by the host to exert bene cial properties, and fecal SCFAs represent non-absorbed SCFAs, so it is reasonable to believe that Sta enhances the ability of BBR promoting SCFAs absorption, resulting in better effects on intestinal barrier integrity, in ammation alleviation, and glucoses metabolism.Meanwhile, it is not excluded that fecal SCFAs reduction induced by BBR with Sta treatment is attributed to lessened diversity and richness of gut microbiota and the subsequent reduction of SCFAs generation.Further investigations on the production, absorption, and excretion of SCFAs due to treatment with BBR with Sta are warranted.
In contrast to most studies on the correlation between SCFAs and gut bacteria, we found that fecal SCFAs are negatively correlated with bene cial bacteria-Bacteroidaceae and Akkermansiaceae, and positively correlated with pathogenic bacteria-Lachnospiraceae.Nevertheless, there are several reports that support our results [39,40].These controversial results may be explained by different models of disease, sample sources of SCFAs, and environment factors.To elucidate the effects of SCFAs and the contradictory ndings about the relationship of SCFAs and intestinal microbiota in diabetes, more efforts are desired.
Notably, the bene cial effects of BBR combined with Sta on T2DM coincides with the formulation thought of ancient Chinese formula-Qianjin Huanglian pill.Our results may provide more evidence on the effect and mechanism of the Qianjin Huanglian pill against diabetes, as well as further clarify the rationality and scienti c basis of the formula.

Conclusion
In

Declarations Figures
Berberine Mice were fed a high-fat diet (45% of energy from fat; Research Diets, USA).Animal experiments were performed in accordance with the "3R" principles and guidelines for laboratory animals (GB14925-2001 and MOST 2006a) established by the People's Republic of China, and approved by the Institutional Animal Care and Use Committee of Institute of Materia Medica (Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China).

Figure 6
Figure 6 summary, Sta as a potential prebiotic enhances the advantageous actions of BBR against T2DM in KKAy mice.And BBR combined with Sta is more effective in glycemic control, in ammation attenuation, and gut integrity maintenance by modulating gut microbiota composition and SCFAs levels, thereby providing a novel strategy for the treatment of T2DM.The speci c mechanism of the combination treatment regulating intestinal microbial composition is unclear and requires further investigation.