Short Chain Fatty Acids Protect the Cognitive Function of Sepsis Associated Encephalopathy Mice via GPR43

A total of 55 male adult C57BL/6 mice (2–3 months of age, 20–25 g) were purchased from the animal core facility of Nanjing Medical University and kept in the barrier system. The mice were maintained under standard conditions: 12 h light/dark cycle, 22 ± 1 °C temperature, 52 ± 2% humidity, free access to food and water. The animal use protocol had been reviewed and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-2109048).

Cecal ligation and puncture in rodents is considered as the gold standard in sepsis research (). The model was established as previously described (). Mice were anesthetized with sodium pentobarbital (40 mg/kg body weight, i.p.). After shaving and disinfection, a midline incision (1.5–2 cm) was made 1.5 cm below the xiphoid to gain access to the peritoneal cavity. The cecum was ligated at midpoint using 3–0 silk thread, then punctured two times at the midway between the ligation and cecal tip, and squeezed gently to empty the fecal contents into the peritoneal cavity. The ligated and punctured cecum was replaced back into the abdominal cavity before closing the peritoneum, abdominal muscle layer, and skin wound. In sham surgery, exteriorized cecum was replaced back into the abdominal cavity without ligation and puncture. Post-operatively, all mice were injected prewarmed (37 °C) sterile .9% saline (1 ml/mouse) subcutaneously to prevent hypovolemia. ^{17 18}

The mice were divided into four groups randomly: sham group (= 10), CLP group (= 15), CLP+SCFAs group (= 15), and CLP+SCFAs+GLPG0974 group (= 15). More mice were assigned to the last three groups to account for the expected mortality of CLP surgery (). Several mice died during the experiment and the final sample size of each group was as follows: sham group (= 10), CLP group (= 10), CLP+SCFAs group (= 12), and CLP+SCFAs+GLPG0974 group (= 10). In the CLP+SCFAs+GLPG0974 group, SCFAs (acetate: propionate: butyrate at a ratio of 3: 1: 1) at 500 mg/kg body weight were administrated intra-gastrically two times a day for 7 consecutive days before CLP surgery, and GLPG0974 at 1 mg/kg body weight were given every 3 days for 24 days by oral gavage right before administration of SCFAs (,). Saline was administrated as vehicle. In the CLP+SCFAs group, CLP surgery and SCFAs were given as described above, and an equal volume of saline was administered instead of GLPG0974. In the sham group and CLP group, only saline pretreatment was given before sham or CLP surgery. Seven days after surgery, fecal samples were collected for microbiota composition and SCFA analysis from 6 mice in each group randomly. Behavioral test was applied to assess cognitive impairment at the same time. After that, the mice were sacrificed and their brains were harvested for further experiment (). SCFAs (acetate, propionate, and butyrate) were purchased from Aladdin, China. GLPG0974 were purchased from Tocris, United Kingdom. n n n n n n n n ^{19 15 20} Figure 1

Spatial learning and memory were assessed by the Morris water maze (MWM) test. A pool of opaque water (100 cm in diameter and 25 cm in depth, 23°C) with a hidden platform (10 cm in diameter) 1 cm below water surface at the center of one quadrant, and a video tracking and analyzing software (ANY-maze, Stoelting, USA) was used.

Between day 7 to day 11 post-operatively, mice were put through MWM 4 times on each training day, starting from a different quadrant each time, with an interval of 20 min between trials. Mice were consistently introduced facing the pool wall. A maximal latency of 60 s was given to reach the hidden platform. Those that failed to reach the platform within 60 s were manually guided to it. After staying on the platform for 10 s, the mice were dried and transferred to the cage. The latency to the platform and path taken were recorded for each mouse. If the mouse failed to reach the platform within 60 s, the latency was recorded as 60 s.

A probe trial was performed on day 12 post-operatively without the submerged platform. The time spent in the target quadrant and platform crossovers were recorded.

A total of 1,000 mg fecal pellets were collected in sterile tubes for each mouse. Microbiota DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, USA) and applied to amplification of V3–V4 regions of 16S rDNA. PCR amplification of the bacterial 16S rRNA genes V3-V4 region was performed using the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After individual quantification, amplicons were pooled in equal amounts, and pair-end 2 × 250 bp sequencing was performed using the Illlumina MiSeq platform with MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd., (Shanghai, China).

Microbiome bioinformatics were performed with QIIME2 2019.4. Briefly, raw sequence data were demultiplexed using the demux plugin followed by primers cutting with cutadapt plugin (). Sequences were then quality filtered, denoised, merged, and chimera removed using the DADA2 plugin (). Non-singleton amplicon sequence variants (ASVs) were aligned with mafft and used to construct a phylogeny with fasttree2 (,). A total number of 867,552 clean reads were obtained. Taxonomy was assigned to ASVs using the classify-sklearn naïve Bayes taxonomy classifier in feature-classifier plugin against the SILVA Release 132 Database (). ^{21 22 23 24 25}

Gas Chromatography-Mass Spectrometer (GC-MS) analysis was used to quantify SCFAs in fecal samples. The sample preparation and GC-MS analysis were performed as described previously (,). Twenty milligrams of fecal samples were accurately weighed and placed in a 2 ml EP tube. One milliliter of phosphoric acid (.5% v/v) solution and a small steel ball were added to the EP tube. The mixture was grinded for 10 s three times, then vortexed for 10 min and ultrasonicated for 5 min. The mixture was centrifuged at 12,000 r/min at 4°C, with 0.1 ml of supernatant added at 10 min. Then, 0.5 mL MTBE (containing internal standard) solution was added. The mixture was vortexed for 3 min and ultrasonicated for 5 min. The mixture was further centrifuged at 12,000 r/min for 10 min at 4°C. The supernatant was collected and used for GC-MS analysis. Agilent 7890 B gas chromatograph coupled to a 7000 D mass spectrometer with a DB-FFAP column (30 m length × .25 mm i.d. × .25 μm film thickness, J&W Scientific, USA) was employed for GC-MS analysis of SCFAs. Helium (flow rate 1.2 ml/min) was used as carrier gas. A 2 μL injection was made in the split mode. The oven temperature was held at 90°C for 1 min, raised to 100°C at a rate of 25°C/min, then raised to 150°C at a rate of 20°C/min, held for .6 min, raised to 200°C at a rate of 25°C/min, then held for .5 min after running for 3 min. All samples were analyzed in multiple reaction monitoring mode. The injector inlet and transfer line temperature were 200°C and 230°C, respectively. ^{26 27}

The hippocampus samples from 6 mice in each group were collected after the behavioral tests, and the level of IL-1β, IL-6, and TNF-α in hippocampus was detected by mouse IL-1β, IL-6, or TNF-α ELISA kits according to the instructions. A monoclonal antibody specific for mouse IL-1β, IL-6, or TNF-α was briefly coated onto the microplates. Wells were incubated for 2 h at room temperature with test samples (hippocampus tissue) and washed five times. Then, 100 μL of mouse IL-1β, IL-6, or TNF-α conjugate was added to each well and incubated further for 2 h. The washing was repeated two times. Wells were then incubated in 100 μL of substrate solution for 30 min and stopped with stop solution (100 μL). Determination of the optical density of each well was set at 450 nm and corrected at 570 nm. A standard curve was constructed using various dilutions of TNF-α, IL-6, and IL-1β standard preparation. The levels of cytokines were calculated according to standard curves. Three replicates were used for ELISA plates to reduce errors. Mouse IL-1β, IL-6, and TNF-α ELISA kits were purchased from Abcam, United States.

All data were expressed as the mean ± standard error of the mean (SEM). Between-group comparisons were analyzed using one-way ANOVA followed by LSDtest for multiple comparisons on normally distributed data. Otherwise, non-parametric Kruskal-Wallis was used. Data from Morris water maze training were analyzed using two-way ANOVA, followed by LSD post hoc test for multiple comparisons (SPSS 20.0 software). The Kaplan-Meier method was used to estimate the survival rate, which was compared by the log-rank test. A value of< 0.05 was considered significant. post hoc p

Sequence data analyses were mainly performed using QIIME2 and R packages (v3.2.0). After rarefaction at 6,000 reads per sample, alpha-diversity metrics were calculated in QIIME2. The linear discriminant-analysis effect size (LEfSe) was further used to identify the dominant bacteria taxa in four groups with the default parameters using R packages (v3.2.0) (). ²⁸

Short chain fatty acids, as a class of key bacterial metabolites, show anti-inflammatory effect in many diseases. In this study, six major SCFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) were measured in fecal samples of the four groups (= 6). The levels of acetic acid (,< 0.001) and propionic acid (,= 0.003) were significantly decreased in the CLP group compared with the sham group, while the levels of butyric acid, isobutyric acid, valeric acid, and isovaleric acid were not influenced (). SCFAs pre-treatment significantly increased the levels of acetic acid (,< 0.001), propionic acid (,= 0.020), and valeric acid (,= 0.002) in CLP+SCFAs group compared with CLP group, while no statistical differences were found in other kinds of SCFAs (). No significant difference was shown between CLP+SCFAs group and CLP+SCFAs+GLPG0974 group (). n p p p p p Figure 2A Figure 2B Figures 2C-F Figure 2A Figure 2B Figure 2E Figures 2C,D,F Figure 2

Gut microbiota was analyzed among the four groups (= 6). The results of alpha diversity demonstrated that chao1 (,= 0.026), Simpson (,= 0.02) and Shannon (,= 0.017) index were significantly different in the CLP group compared with the sham group. n p p p Figure 3A Figure 3B Figure 3C

In order to further study differences in gut microbiota composition, the top 20 bacteria taxa on genus level with the highest relative abundance were analyzed (). Among them, SCFAs-producing bacteria such as(,= 0.037),(,= 0.002) and(,= 0.002) was significantly reduced in the CLP group compared with the sham group.(,= 0.002) was significantly increased in the CLP+SCFAs group compared to the CLP group, while no significant differences were found in() and(). The levels of SCFAs-producing bacteria were not influenced by GLPG0974 as no significant differences were found between CLP+SCFAs group and CLP+SCFAs+GLPG0974 group (). Figure 3D Figure 3E Figure 3F Figure 3G Figure 3E Figure 3F Figure 3G Figures 3E-G Allobaculum p Bacteroides p Bifidobacterium p Allobaculum p Bacteroides Bifidobacterium

Furthermore, LEfSe analysis was used to identify the dominant bacteria taxa in different groups. A total of 39 bacteria taxa with statistically significant and biologically consistent differences were found (). The phylum, class, genus, etc. were significantly enriched in the sham group. While the family, genus, genus, etc. were most likely to explain the differences between the CLP group and the other mice. In the CLP+SCFAs group, phylum, class, order, etc. were significantly increased. In the CLP+SCFAs+GLPG0974 group, the family, genus, phylum, etc. were uniquely enriched. Among them,, andwere more abundant in the sham, CLP+SCFAs or CLP+SCFAs+GLPG0974 groups. Figure 4 Firmicutes Bacilli Allobaculum Burkholderiaceae Burkholderia Dehalobacterium Actinobacteria Actinobacteria Bifidobacteriales Bacteroidaceae Bacteroides Proteobacteria Allobaculum, Bifidobacterium Bacteroides

Morris water maze was performed to analyze the cognitive functionof each group at 7 days post-operatively.

Two mice died due to gavage in the CLP+SCFAs+GLPG0974 group. The post-operative 7-day survival rates of mice in the sham, CLP, CLP+SCFAs, and CLP+SCFAs+GLPG0974 groups were 100.0% (10/10), 66.7% (10/15), 80.0% (12/15), and 76.9% (10/13), respectively (). Two mice in the CLP+SCFAs group were excluded from MWM due to poor wound healing. No mortality was reported during behavioral testing. Figure 5A

In the MWM, there was no significant between-group difference in latency and average speed in the training phase (), which indicates that all mice had learnt to achieve the exercise goal after 5 days of training, and mobility was not affected by sepsis. However, in the probe trial, the CLP group spent less time in the target quadrant (,< 0.001) and had fewer crossings in the target quadrant (,< 0.001) than the sham group. In contrast, the CLP+SCFAs group spent more time in the target quadrant (,< 0.001) and had more crossings (,< 0.001) in the target quadrant compared with the CLP group, and GLPG0974 reversed these changes (,< 0.001,,= 0.001). Figures 5B,C Figure 5D Figure 5E Figure 5D Figure 5E Figure 5D Figure 5E p p p p p p

The levels of several inflammatory cytokines in the hippocampus were measured to evaluate neuro-inflammation. Compared with the sham group, the levels of IL-1β (,< 0.001), IL-6 (,< 0.001) and TNF-α (,< 0.001) were significantly increased in CLP group. Conversely, the levels of IL-1β (,< 0.001), IL-6 (,= 0.006) and TNF-α (,< 0.001) were significantly lower in the CLP+SCFAs group than those in the CLP group. GLPG0974 reversed the anti-neuroinflammatory effect of SCFAs, by increasing IL-1β (,= 0.038), IL-6 (,= 0.002), and TNF-α (,= 0.002) significantly in the CLP+SCFAs+ GLPG0974 group compared with the CLP+SCFAs group. Figure 6A Figure 6B Figure 6C Figure 6A Figure 6B Figure 6C Figure 6A Figure 6B Figure 6C p p p p p p p p p