Gut microbiota-derived gamma-aminobutyric acid from metformin treatment reduces hepatic ischemia/reperfusion injury through inhibiting ferroptosis

Hepatic ischemia/reperfusion injury (HIRI) is a common and inevitable factor leading to poor prognosis in various liver diseases, making the outcomes of current treatments in clinic unsatisfactory. Metformin has been demonstrated to be beneficial to alleviate HIRI in recent studies, however, the underpinning mechanism remains unclear. In this study, we found metformin mitigates HIRI-induced ferroptosis through reshaped gut microbiota in mice, which was confirmed by the results of fecal microbiota transplantation treatment but showed the elimination of the beneficial effects when gut bacteria were depleted using antibiotics. Detailedly, through 16S rRNA and metagenomic sequencing, we identified that the metformin-reshaped microbiota was characterized by the increase of gamma-aminobutyric acid (GABA) producing bacteria. This increase was further confirmed by the elevation of GABA synthesis key enzymes, glutamic acid decarboxylase and putrescine aminotransferase, in gut microbes of metformin-treated mice and healthy volunteers. Furthermore, the benefit of GABA against HIRI-induced ferroptosis was demonstrated in GABA-treated mice. Collectively, our data indicate that metformin can mitigate HIRI-induced ferroptosis by reshaped gut microbiota, with GABA identified as a key metabolite.


Introduction
6][7][8] Unfortunately, current treatments are not satisfactory due to the complicated pathogenesis of HIRI.Recently, a novel theory relating to the gut-liver axis has been well accepted, demonstrating that gut microbiota is vital for liver diseases, however, there is no strategy targeted this theory to alleviate HIRI in clinical practice.
The liver links with the intestine by portal circulation which allows the transfer of gut-derived products, not only nutrients, but also microbial metabolites and components. 9As an important part of the gut-liver communication, the intestinal mucosal and vascular barriers limit the systemic dissemination of microbes and toxins while allowing nutrients to access the circulation and reaching the liver. 10Additionally, it is generally believed that liver injury can provoke intestinal mucosal damage and inflammation, further resulting in gut dysbiosis, which reversely induces a secondary attack on the liver. 11Microbial agents, such as synbiotics and probiotics, have demonstrated to attenuate different liver diseases in animal studies, 12,13 indicating that modulating gut microbes potentially prevents HIRI in clinical practice.
5][16][17] As evidences shown, the gut with high concentration of metformin become an important action site to reshape microbiota. 18According to Sun et al., 19 metformin was proven to decrease the abundance of the B. fragilis species and to increase the levels of bile acid and glycoursodeoxycholic acid in the gut, which inhibited intestinal farnesoid X receptor against obesity-related metabolic dysfunction in mice.Moreover, it is also demonstrated that metformin could attenuate sepsis-related liver injury by increasing the proportion of Bifidobacterium, Muribaculaceae, Parabacteroides distasonis, and Alloprevitella in rats. 20As for the HIRI, metformin has also shown the protective effect, 21 but the detailed role of gut microbiota remains unclear.
Although metformin showed protective effects on HIRI, the role and distinctions of reshaping microbes have yet to be determined.In the present study, we sought to elucidate that gammaaminobutyric acid (GABA)-producing bacteria played a critical role for the protective effects of metformin against HIRI by using 16S rRNA and metagenomic sequencing to find the different features of gut microbiota.The finding was reconfirmed by the results of mice that were treated with metformin-reshaped fecal microbiota or oral antibiotics administration.Furthermore, GABA was found to show a protective effect on HIRI by intraperitoneally injection into mice in advance, while an increase of microbial GABA was observed in both mice and human experiments after metformin treatment.Evidenced by the improvement of HIRI, we hypothesize that metformin-reshaped gut microbiota elicits greater therapeutic responses for liver diseases and serve as a promising targeted therapeutic.

Metformin significantly mitigates HIRI and reshapes gut microbiota in mice
In order to evaluate the protective effect of metformin on HIRI, survival experiment was employed in mice.The survival rate of HIRI mice increased from 41.67% to 90.00% after metformin intervention (Fig. 1a), accompanied by hepatic morphological and histopathological improvement including the recovery of liver pathological structure, the decrease in red blood cell aggregation and consumption of liver glycogen (Fig. 1b), as well as the decrease in serum activity of ALT and AST (Fig. 1c).Qualification of MDA and GSH concentrations in the mice liver demonstrated that the presence of metformin significantly attenuated HIRI-induced oxidation (Fig. 1d).
In order to clarify the critical role of gut microbiota in the pleiotropic actions of metformin, [22][23][24] fecal samples were collected from the mice to perform 16S rRNA sequencing.It was found that the gut microbiota was substantially modulated in IR + metformin (Met) group, showing decreased abundance (alpha diversity) and reshaped distribution (Fig. 1e).Next, as the microbiota-gut-liver axis theory indicates, 25 HIRI-induced dysfunction of the gut barrier may aggravate liver damage by disrupting the gut microbiota.H&E staining of the colon and ileum, mainly involved in gut barrier dysfunction, showed that the density and integrity of intestinal mucosa were strikingly reduced by HIRI, which was restored by metformin treatment (Fig. 1f).
The immunofluorescence analysis of colon tissues showed that the decreased tight junction protein ZO-1 and Occludin in HIRI-treated mice were reversed by metformin (Fig. 1g and 1h).
Similarly, western blot analysis of Occludin in colon (Fig. 1g) and the mRNA levels of Ocln, Tjp1, Cldn7, F11r and Igsf5 in ileum of HIRI mice were increased with metformin administration (Fig. 1i).The inflammatory cytokine Il6, Il1b and Il18 were significantly upregulated in the ileum and colon received HIRI and were reduced by metformin treatment (Fig. 1j).In addition, the serum FD-4 level in IR group was cut down by metformin (Fig. 1k).
These results suggested that metformin plays a critical role in restoring gut barrier function and alleviating HIRI.

Metformin-reshaped fecal microbiota attenuates HIRI
In order to further confirm the role of metformin in reshaping the gut microbiota, microbes were transplanted into mice before inducing ischemia reperfusion (IR).As expected, the survival analysis of mice in the IR + fecal microbiota transplantation (FMT) group showed a similarly protective efficacy as the IR + Met group, as indicated by the significantly improved survival rate of IR mice (Fig. 2a).This finding was validated by ALT and AST levels as well as H&E, PAS and DHE staining (Fig. 2b and 2e).FMT treatment could restore the GSH level but reduce the accumulation of MDA in hepatic tissues (Fig 2c and 2d).Furthermore, a decreased alpha diversity was observed in the FMT group, similar to IR + Met group (Fig. 2f).Meanwhile, the attenuation of gut damage was showed by H&E staining, and the improvement in tight junction proteins ZO-1 and Occludin, and decreased HIRI-induced Il1b, Il6, and Il18 (Fig. 2g-2i).Serum FD-4 level showed a sharply decrease in IR + FMT group (Fig. 2j).To further identify the function of gut microbes, experiments were designed, and combination treatment of antibiotics (1 mg/mL penicillin sulfate, 1 mg/mL neomycin sulfate, 1 mg/mL metronidazole and 0.16 mg/mL gentamicin) and metformin were employed for 1 week before IR treated.As results showed, the ALT, AST and MDA detection in control group treated with Abx showed no obvious change to control group, indicating that the Abx had no influence on the liver.The survival rate of IR + Met + antibiotics (Abx) group was greatly reduced, while the levels of ALT, AST, MAD and GSH were completely opposite to IR + Met group (Fig. 2k).The results confirmed that gut microbiota is critical for the effect of metformin against HIRI.

FMT alleviates HIRI-induced ferroptosis through reshaped fecal microbiota
Since the reperfusion-induced excessive oxidative stress takes place in iron-rich hepatocytes, 26 experiments were designed to observe ferroptosis in HIRI.The ferroptosis inhibitor, DFO, significantly ameliorated the liver injury, suggesting that ferroptosis contributes to HIRI (sFig.1).Hence, to find out the relation, ferroptosis-related indicators were also monitored in both IR + Met and IR + FMT groups.The increased accumulation of Fe after HIRI was significantly cut down by metformin and FMT treatment (Fig. 3a).The quantitative Real-time PCR (qRT-PCR) analysis showed that the HIRI-induced upregulation of ferroptotic parameters, Acsl4, Slc7a11, Slc39a14 and Ptgs2, were significantly decreased by metformin and its reshaped gut microbiota, respectively (Fig. 3b-3e).The results of western blotting further confirmed the anti-ferroptosis of metformin and FMT by the remarkable reduction of ACSL4, TFR1, VDAC1, VDAC2 and VDAC3 levels and the apparent increase of antiferroptotic FTH1 and Xct levels (Fig. 3f), The results were further consolidated by lipid ROS staining and transmission electron microscopy (TEM) analysis (Fig. 3g).Additionally, transcriptome data on genes associated with antiferroptosis showed an increase in both IR + Met and IR + FMT groups, whereas proferroptic genes showed a decrease, when compared with IR group (Fig. 3h).Collectively, the results suggested that FMT performed protection against HIRI by inhibiting ferroptosis.

Metformin induces GABA-producing gut microbiota
To elucidate the molecular mechanisms through which pathway participates metformin-treated IR injury, we analysed gene expression profiles of each group mice.Transcriptome sequencing analysis revealed that 9697 genes were in common among four groups (sfig.6).Therefore, we used these common genes for KEGG analysis, showing that similar mRNA changes are mainly concentrated in the three top pathways: lipid metabolism, carbohydrate metabolism, and amino acid metabolism (Fig 4a).Given the close relevance between lipid metabolism and ferroptosis (sFig.2), and the fact of carbohydrate metabolism is a primary way to metabolize amino acids, 22 species of amino acid were detected in liver tissues using HPLC-MS/MS for further identification of key metabolites involved in the role of metformin against HIRI-induced ferroptosis.It was found that only GABA level is significantly increased by metformin treatment and FMT treatment (sFig.3), further verifying by the data of ELISA detection (Fig. 4b).Moreover, it was observed that the genus of Bacteroides had a significant increase based on the 16s rRNA gene sequencing of metformin-treated mice microbes.qRT-PCR analysis of eukaryotic GABA synthesis key enzymes including Aldh5a1, Abat, Gad1 and Gad2 in liver tissues, were not upregulated by metformin (Fig. 4d), which was also confirmed by western blotting detection of GAD1 and GAD2 in liver (Fig. 4f).Interestingly, the fecal GABA concentration was shown a significant increase in metformin-treated mice, which was copied by the IR + FMT group, but eliminated by antibiotics treatment (Fig. 4e).In order to further identify the increased GABA originates from gut microbiota, two key enzymes of prokaryotes GABA synthesis, GAD and PAT, were detected on DNA level, finding that both of them are significantly increased in the feces from IR+Met and IR+FMT groups (Fig. 4g and h).In order to clarify the specific effects of metformin on microbiota, given the big safety margin, healthy volunteers were recruited for a 1 week of daily oral 500mg dose of metformin trial.Fecal samples were collected before and after oral administration of metformin for metagenomic analysis.Detailedly, the species of Bacteroides containing Bacteroides thetaiotaomicron, Bacteroides unifomis, and Bacteroides salyersiae, were enriched in human gut after metformin administration (Fig. 4i).Excitingly, the increase of GABA production, and the expression of GAD and PAT were shown in the results of fecal experiments in vitro and in vivo, accompanied by the decreased glutamic acid (Glu), a key substrate for gut microbiota to synthesize GABA (Fig. 4j and k).Besides, putrescine, another substrate of GABA synthesis, was not found in primary culture medium but appeared after 24h fermentation and then was consumption later in the anaerobic culture of human feces experiment.(Fig. 4l).Taken collectively, GABAproducing gut microbiota was upregulated by metformin.

GABA is the critical metabolite of metformin-reshaped gut microbiota against HIRI-induced ferroptosis
The animal experiment was designed to further observe the effects of GABA on HIRI-induced ferroptosis (Fig. 5a).As expected, the data showed that GABA treatment reduced histological damage and liver glycogen consumption (Fig. 5b).Serum liver function detection showed that ALT and AST activities were dramatically depressed after GABA treatment (Fig. 5c).The decreased MDA and Fe as well as increased GSH were observed in further determination, confirming the protection of GABA on liver (Fig. 5d).The results of western blotting showed the anti-ferroptosis effect of GABA treatment by the significant reduction in ACSL4, TFR1, VDAC1,2,3 and increase of anti-ferroptotic FTH1 levels, which had the same attenuating effect as metformin and FMT treatment (Fig. 5e).In addition, the clustergram showed GABA upregulated the genes of antiferroptosis and depressed the proferroptic ones, which was similar to the effect after metformin treatment (Fig. 5f).In summary, GABA treatment reduced HIRIinduced ferroptosis.

Discussion
Metformin has been well researched for treating various liver diseases due to its multifarious effects.Notably, it has been well documented that oral administration of metformin lead to therapeutic effects on HIRI, 27 leaving a thought-provoking question in its underlying mechanism.In the current study, the changes in the gut microbiota altered by metformin are demonstrated to be necessary and sufficient for conferring liver injury protection.Moreover, it has been confirmed that metformin attenuated HIRI by the increased GABA from reshaped gut microbiota, through anti-ferroptosis.
Increasing evidence shows that the gut microbiota is critical for the pleiotropic actions of metformin. 25,28,29 Iterestingly, metformin was previously found to have a much higher concentration in the gut lumen than other organs, 18 as also confirmed by the current study.In fact, that concentration favoured the action of metformin on reshaping gut microbes.Despite the distinct heterogeneity of metformin-reshaped gut microbiota in recent research, Bacteroides with a high abundance of gut microbes was reported to be reduced after metformin treatment in studies of hyperlipidemia and diabetes, 19,30 which other studies vary from for the different action of this bacteria. 31,32 n our study, we used a disease model of HIRI, which may have unique characteristics compared to other disease models.It is possible that the specific disease model influenced the response of the gut microbiota.Additionally, the starting microbiota of the recipients and the characteristics of the donor microbiota used for FMT could also play a role in the disparity.Our findings showed an increase abundance of the genus of Bacteroides after metformin treatment supported by the data of mice and healthy volunteers.Detailedly, the increased Bacteroides especially Bacteroides thetaiotaomicron, Bacteroides unifomis, and Bacteroides salyersiae were observed in the human gut after metformin administration.The enriched Bacteroides theaiotaomicron, a glutamate-fermenting commensal, was reported to be decreased in obese individuals and was inversely correlated with serum glutamate concentration. 33Therefore, Bacteroides theaiotaomicron might be the potential bacterium regulating of GABA production.Little research has been done on Bacteroides unifomis and Bacteroides salyersiae, needing a further exploration of their characteristics in the future.
Notably, Phascolarctobacterium, coexistly with Bacteroides thetaiotaomicron, 34 was reported as an emerging probiotic which showed a significant increase in our study.Taken collectively, those reshaped bacteria after metformin treatment may be responsible for attenuating HIRI.
Increased GABA was shown from the metformin reshaped gut microbiota for the first time, verified by our results of feces from mice and humans.As the critical metabolite of bacteria against acid stress, GABA was extensively produced by gut microbes, especially the species with strong capacity of acid resistance.Interestingly, our analysis of the sequences in PUBMED Gene bank found that those three species of Bacteroides, Bacteroides thetaiotaomicron, Bacteroides unifomis, and Bacteroides salyersiae, which belong to metformin reshaped microbiota, contain the key enzyme genes of GABA synthesis GAD and PAT (sFig 4), further confirmed by the qRT-PCR result of human fecal DNA in vivo and in vitro experiment.
Specifically, the species with the strong ability of producing GABA such as Bacteroides theaiotaomicron, Bacteroides unifomis and Bacteroides salyersiae were increased in metformin-treated volunteers.Some studies were published to elucidate the mechanism of metformin regulating bile acids and folate metabolite by reshaped gut microbiota, however, there is no report about how metformin to affect GABA synthesis of gut microbes.The data from anaerobic cultured human feces showed that metformin promoted the increase of GAD and PAT, and GABA level along with the substrate consumption of glutamate and putrescine in a time-dependent manner.
GABA production was reported to be critical against acid stress for microbiota to survive and preserve metabolic activity, especially in the human gut with a lower pH than other animals. 35ns benefit from bacteria-derived GABA.Peripheral GABA was first demonstrated in intestinal flora in 1981 and further confirmed by recent studies. 36In addition to the important role in the central neural system and immune system, GABA showed a protective effect in different acute liver injury models. 37,38 ublished papers have shown that GABA performs beneficial effect through binding to the GABA receptors or promoting histone acetylation. 39,40 ata from GABA-treated mice showed protective effects against HIRI-induced ferroptosis.
However, further studies are required to examine the exact molecular mechanisms of GABA regulating ferroptosis.
In conclusion, we have demonstrated metformin-reshaped gut microbiota performs therapeutic responses for HIRI.Gut microbiota has been obviously modified while receiving oral metformin treatment, suggesting that metformin-reshaped gut microbiota has enhanced intestinal mucosal function so that reduce harmful substances to the liver, which most efficiently evade damage.Specifically, GABA producing bacteria were significantly regulated after metformin administration.Demonstrated for the first time, we showed that the increased GABA is a key metabolite of gut microbiota for metformin to mitigate HIRI.These results offer a unique therapeutic strategy, with optimized gut microbiota, that can serve as a target for metformin treating liver diseases.

Animals
Six-to-eight-week-old specific pathogen-free male C57BL/6 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co. Ltd. (Beijing, China).All mice were housed under controlled temperature and humidity conditions with a 12-h light-dark cycle and free access to food and water.The mice were fasted overnight before the experiments.All animal experiments were approved by the Institutional Animal Care and Use Committee of the Experimental Animal Centre of Wenzhou Medical University (wydw2021-0348).

Healthy volunteers
All research was conducted in accordance with both the Declarations of Helsinki and Istanbul, all research was approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University (2022-K-08-01), and written consent was given in writing by all subjects.

Hepatic ischemia/reperfusion (IR) mouse model
A mouse model for HIRI was established as previously described. 3Briefly, mice were anaesthetised with pentobarbital sodium.A noninvasive microvascular artery clip was placed on the left branches of the hepatic artery and portal vein for 30 min, and the clip was removed for reperfusion for 6 h.During the study period, body temperature was maintained at 37 °C using a heating lamp.

Experimental design
Mice were randomly divided into the several groups.In general, the arterial and portal vessels to the cephalad lobes were clamped for 30min and reperfusion for 6 h.No vascular occlusion in sham-controlled mice.In the treatment groups, animals were treated with metformin (1 mg/mL), metformin modified fecal microbiota transplantation or metformin (1 mg/mL) mixed with antibiotics (1 mg/mL penicillin, 1 mg/mL neomycin sulfate, 1 mg/mL metronidazole and 0.16 mg/mL gentamicin), in the drinking water for 1 week.Specifically, FMT-treated mice were fed with washed metformin-treated mice feces, which had been detected by HPLC showing that there was no metformin left in the feces.Other two groups of mice were infused at 1 h prior to the onset of liver ischemia with a single dose of deferoxamine (DFO) (20 mg/kg i.p.) or GABA (100 mg/kg i.p.), dissolved in PBS.Mice were sacrificed after reperfusion; liver and serum samples were collected for analysis.

Hematoxylin-eosin (H&E) and periodic acid-schiff (PAS) staining
For histopathological examination, liver and intestinal tissues were fixed in 4% paraformaldehyde overnight.Selected tissue blocks were processed using a routine overnight cycle in a tissue processor.Tissue blocks were then embedded in wax and serially sliced into 5μm sections.H&E and PAS staining performed as instructions provided by the reagent manufacturer (Solarbio, G1120, G1281).Visualization and images were obtained under an optical microscope (Nikon, Tokyo, Japan).

Transmission electron microscopy (TEM) analysis
After reperfusion, the mice were euthanized, and the livers were excised and washed with precooled PBS (pH 7.4).Part of the liver was then removed and incubated overnight in 0.1 M PBS (pH 7.4) containing 2.5% glutaraldehyde.The target tissues were cut into 50-µm-thick sections using a vibratome.Selected areas of the livers were postfixed in 1% osmium tetroxide for 1 h, dehydrated in a graded ethanol series and embedded in epoxy resin.The polymerisation was performed at 80 °C for 24 h.Ultrathin sections (100 nm) were cut, stained with uranyl acetate and lead citrate and viewed under a JEM2000EX TEM (JEOL, Tokyo, Japan).Five fields were randomly selected for each sample to examine mitochondria with ferroptosis features.

Quantitative Real-time PCR (qRT-PCR) analysis
Total RNA was extracted from liver and intestinal tissues using TRIzol (Yamei, YY101).

Western blotting
Total protein samples were extracted from tissues using RIPA lysis buffer (Yamei, PC101).

High-performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS) detection of metformin and liver amino acids
Sample separation was performed using a ZORBAX Eclipse XDB-C18 column (4.6 × 150 mm, Agilent, USA) using an injection volume of 5 μL.Metformin detection conditions are as follows: column temperature of 30°C, one mobile phase is NaH 2 PO 4 (Sigma, V900060) containing 5 mmol/L sodium dodecyl sulfonate (Sigma, V900859) with pH 3.5 while the other mobile phase is acetonitrile (Aladdin, A104440).The metformin content in each sample was measured by HPLC (Agilent 1260, USA).Amino acid detection conditions are as follows: column temperature of 40°C, mobile phase A of 10% methanol/water (containing 0.1% formic acid), mobile phase B of 50% methanol/water (containing 0.1% formic acid) and flow rate of 0.4 mL/min.Mass spectrometry was performed using a triple quadrupole mass spectrometer with an ESI source in negative ionisation mode.High-purity nitrogen was used as the nebulising and drying gas.Quantification was performed in multiple reaction monitoring mode.

16S rRNA sequencing
16S rRNA amplicon sequencing was performed at Chunlab Inc. (Seoul, Korea) with MiSeq system (Illumina).Briefly, for preparation of MiSeq library amplicons, target gene (16S rRNA V3-V4 region) was amplified using338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') primers, using an ABI GeneAmp 9700 PCR thermocycler (ABI, CA, USA).The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions and quantified using a Quantus Fluorometer (Promega, USA).Normalization was performed the counts of individual OTUs in a sample by dividing the total counts of all OTUs within that sample followed by a multiplication by resulting in relative abundance expressed.

Metagenomic sequencing
Total genomic DNA was extracted from human fecal samples as mentioned in 16S rRNA sequencing.DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction.Paired-end library was constructed using NEXTflexTM Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA).
Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt-end of fragments.Paired-end sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd.(Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer's instructions (www.illumina.com).

Statistical analysis
GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA) was used for statistical treatment.Experimental data were shown as the mean ± SD.Two-tailed unpaired Student's ttest and one-way ANOVA with Tukey's correction were used for all comparisons of mice-related experiments.A P value < 0.05 was considered significant.The sample distribution was determined using a Kolmogorov-Smirnov normality test.

Figure 1 .
Figure 1.Metformin significantly mitigates HIRI by reshaping gut microbiota in mice