The effect of Sennoside A on the improvement of lipopolysaccharide -associated encephalopathy through gut microbiota

suggests that the intestinal ora is involved in many neurodegenerative Sepsis can lead to severe intestinal ora and brain dysfunction. this Sennoside A may relieve lipopolysaccharide(LPS)-associated via gut microbiota in


Introduction
Sepsis is one of the leading causes of morbidity and mortality in patients in critical care and acute care wards [1] . Sepsis-induced brain dysfunction is called sepsis-associated encephalopathy (SAE), the mechanisms underlying these complications remain unclear and an effective intervention is lacking [2] . Thus, exploring the pathogenesis of SAE is important to develop more effective prevention and treatment strategies.
Accumulating evidence suggests that the intestinal ora is involved in many neurodegenerative diseases. Ma et al. discovered Bi dobacterium infantis M-63 elevated mental health in individuals with irritable bowel syndrome that developed after a major ood disaster [3] . Lowe et al. found that acute-on-chronic alcohol administration induces alcohol-associated central nervous system and gut in ammation, which could be reduced by gut microbiome intervention [4] . A previous study from our group proved that modulation of the intestinal microbiota in septic rats could alleviate SAE [5] .
Sepsis can lead to severe intestinal ora imbalance. Chen discovered that the structure and composition of intestinal ora change signi cantly in rats with sepsis [6] . Gong determined that gut microbiota imbalance was an important mediator of sepsis-induced liver injury [7] . Beckmann found that burn injury patients had a high risk of sepsis-related mortality, and these patients had signi cant changes in their intestinal microbiome composition [8] . To Modulating this microbiota dysbiosis may be a potential method to improve SAE in septic rats.
Therefore, targeting the gut microbiota with appropriate drugs is hypothesized to be an effective therapy for SAE. Sennoside A, an inactive glycoside in rhubarb, is the major purgative component of rhubarb [9] . Rhubarb is a common traditional Chinese medicine used to regulate enteric function. It is known that rhubarb can rectify an intestinal ora imbalance [10] .Yao discovered that rhubarb can rectify intestinal ora in rats with severe acute pancreatitis [11] . The anti-tumor properties of rhubarb have stimulated research to assess its effect in shaping the gastrointestinal bacterial diversity of young animals when used as a feed additive [10] .
Accumulating evidence supports the nding that Sennoside A and rhubarb can alter intestinal bacterial composition and hence exert multiple pharmacological effects [12] . Nevertheless, it remains unknown whether Sennoside A affects LPS-associated encephalopathy via the gut microbiota. Fecal microbiota transplantation (FMT), as an effective methods to improve the dysbosis of intestinal microbiota, is an effective method in SAE treatment [5] .
To test the hypothesis that gut microbiota alteration may be involved in the pathogenesis of LPS-associated encephalopathy, and may be ameliorated by Sennoside A, LPS-induced sepsis was used, LPS can make "model of acute in ammation", which can be used to study sepsis [13] . Ordinary and germ free SD rats were adopted as a model. The ratio of abnormal EEGs, levels of TNF-α, IL-1β, and IL-6 in the rat cortexes were assessed after Sennoside A administration or FMT. The effects of Sennoside A on intestinal microbiotas were evaluated by analyzing the bacterial V3-V4 regions of 16S rRNA genes by Illumina sequencing and multivariate statistical analysis.

Materials And Methods
Animals Eighty adult male Sprague-Dawley (SD) rats and forty germ free (GF) rats were purchased from Hebei Medical University, Shijiazhuang, China. The protocol was approved by the Ethics Committee of Hebei General Hospital, and all procedures were performed in accordance with the Guideline for the Care and Use of Laboratory Animals from the National Institutes of Health, USA. The animals were housed under a 12-h light/dark cycle in a temperature-controlled room at 24±1°C with free access to food and water. Germ free rats were given free access to germ free food and water (all food, water, bedding and other supplies that were put in the isolator were sterilized in advance) [14] .

Experimental procedures
Eighty ordinary SD rats were randomly divided into four equal groups: Sham treatment group, LPS group (LPS+saline), LPS with Sennoside A treatment group (LPS+SA), LPS with FMT treatment group (LPS+FMT).
Germ free SD rats were randomly divided into two groups: rats given LPS (LPS+GF+saline), and rats given LPS and Sennoside A treatment (LPS+GF+SA). In the LPS+SA group, rats were gavaged with Sennoside A at 25mg.kg -1 every 8 hours; in the sham and LPS +saline groups, rats were given normal saline instead of Sennoside A. For the rats in LPS +FMT group, fecal microbiota transplantation was performed with fresh feces from the healthy donor rats three times a day for 7 days. Germ free rats in the LPS+GF+SA group were gavaged with Sennoside A at 25mg.kg -1 every 8 hours. In the LPS+GF+saline group, rats were given normal saline instead of Sennoside A. Sennoside A and saline were sterilized in advance. Rats in all groups except the sham group received an intravenous injection of 10mg/kg body weight lipopolysaccharide(LPS from Escherichia coli, O111:B4; Sigma-Aldrich Chemie GmbH, Germany) through the caudal vein, while rats in the sham group were given the same volume of saline. After the 7 th day, fecal samples were collected, and 1g of each sample was immediately stored at −80°C until DNA extraction and subsequent use to study microbiota composition with 16S rDNA analysis.

Enzyme-linked immunosorbent (ELISA) assay
The brains were removed, stored at −80°C; the brain cortex samples were collected for TNF-α, IL-1β, and IL-6 detection 48h after LPS or saline administration. The concentrations of TNF-α, IL-1β, and IL-6 were detected by ELISA kits according to the manufacturer's instructions. Standard curves were constructed using various dilutions of manufacturer-supplied TNF-α, IL-1β, and IL-6 standards. The levels of the cytokines were calculated according to the standard curves.

PCR and sequence analysis
Fecal samples were harvested and used for bacterial DNA extraction and sequencing of the V3-V4 hypervariable region in the 16S rDNA gene.

EEG Recordings and analysis
EEGs were recorded at 48h after LPS or saline administration. Standard EEG was performed using a Nihon Kohden manufactured EEG-9100J/K portable digital EEG system. EEG recordings and analysis followed the guidelines of the International Federation of Clinical Neurophysiology.

Statistics analysis
All data are presented as mean±standard error of the mean (SEM) or standard deviation (SD). Statistical analyses were performed using a one-way analysis of variance. In cases of signi cance, a Fisher post hoc test was applied (Statview, SAS, Cary, NA, USA). Correlation between two variances was estimated using linear regression analysis with a Pearson test. The signi cance level was set to P<0.05. Analyses of microbiomes showed shifts in bacterial relative abundances upon FMT and Sennoside A supplementation. As illustrated FIG.2a, at the phylum level, the most abundant microbiota in the gut of LPStreated rats were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. The microbiome composition was altered in the LPS+saline group: there was signi cantly greater abundance of Proteobacteria and signi cantly lower abundance of Firmicutes compared with the sham group. Meanwhile, the abundance of Bacteroidetes was decreased, whereas Actinobacteria was almost unchanged. These values re ect the percentage of the mean relative abundance of this bacterial group in the four groups (not including the germ free groups). Sennoside A supplementation and FMT were found to markedly modify relative percentages of detected phyla when compared to the LPS+saline rats. Sennoside A and FMT supplementation were able to marginally (P=0.00, P=0.00) increase Firmicutes, and Bacteroidetes. Bacterial community composition was similar in Sennoside A and FMT groups compared to the sham group.
As illustrated FIG.2b, at the family level, in the LPS+saline rats, the mean relative abundances of Ruminococcaceae, Clostridiaceae, Lactobacillaceae, Erysipelotrichaceae, Bacteroidaceae, and Eubacteriaceae, were signi cantly reduced compared to the sham-treated rats, meanwhile, the mean relative abundances of Lachnospiraceae, Prevotellaceae, Graciibacteraceae, Acidaminococcaceae, and Acholeplasmataceae were signi cantly increased compared to the sham-treated rats. The mean relative abundances of Ruminococcaceae, Clostridiaceae, and Lachnospiraceae were the most affected bacterial groups after Sennoside A supplementation and FMT; the mean relative abundance of Ruminococcaceae and Clostridiaceae were signi cantly increased compared to the LPS+saline rats, and the mean relative abundance of Lachnospiraceae was signi cantly decreased compared to the LPS+saline rats.
Regarding the genus level, LPS+saline rats had signi cantly reduced levels of Faecalibacterium when compared to sham-treated rats. In contrast, LPS+saline rats had signi cantly increased levels of Alistipes, compared to the sham group. Sennoside A and FMT treatments signi cantly increased the levels of Faecalibacterium when compared to LPS+saline rats, and reduced the abundance of Alistipes compared to the sham group (FIG.3a,3b ).
SAE rats usually present with higher levels of in ammatory cytokines, including TNF-α, IL-1β, and IL-6 in cortex tissues [5] . We measured the levels of these cytokines after the 7 th day of Sennoside A feeding. We determined that the levels of these cytokines were lower in the cortex tissues of Sennoside A-fed rats.

4.Sennoside A improved LPS-associated encephalopathy
Abnormal EEGs indicate the degree of SAE [13] . EEGs showed that in rats treated with LPS there was a remarkable increase of reactivity, excessive theta and delta activity, electrographic seizures (ESZ) and periodic discharges (PD) compared with the sham-treated group. However, these abnormalities were reduced in the groups of rats treated with Sennoside A and FMT. These results suggest that Sennoside A improves brain function of rats with sepsis (Table1). Thus, both Sennoside A and FMT relieved in ammation in the cortex tissues of rats with sepsis.
To further con rm the hypothesis that SAE was affected by the gut microbiome, germ free rats were studied. The ratios of abnormal EEGs were not improved after Sennoside A therapy in germ free rats(Table1). In short, Sennoside A likely exerts its brain protective effects through gut microbiota alteration.

Discussion
This study demonstrated that Sennoside A treatment improved the dysbiosis of intestinal microbiota in LPS-associated encephalopathy rats. Sennoside A supplementation had a substantial impact on gut microbiota diversity and composition at different taxonomic levels. We were particularly interested in the abundance of bacterial species such as Faecalibacterium and Alistipes, which had previously been associated with brain function. The incidence of abnormal EEGs, the cortex levels of TNF-α, IL-1β, and IL-6, were also signi cantly decreased in the Sennoside A group compared with the LPS group.
More interestingly, in GF rats, Sennoside A did not lower the ratio of abnormal EEG, or lower TNF-α, IL-1β, and IL-6 levels in rat cortexes. Abnormal EEG and elevated in ammatory mediators indicate the degree of SAE [14] . The present results support the hypothesis that gut microbiota is involved in the pathogenesis of LPS-associated encephalopathy and that Sennoside A exerts its brain protective effects through gut microbiota alteration.
Other authors have also shown that Sennoside A, the main glycoside in rhubarb, can normalize the gut microbiota. Wei et al., found that Sennoside A could effectively improve the abundance of the intestinal ora, and alleviate Type 2 diabetes symptoms and reduce obesity [15] . Sennoside A restored the gut microbiota pro le, increased short chain fatty acids (SCFAs), improved mucosal structure in the colon and restored the function of the microbiota-GLP1 axis to improve glucose metabolism in obese mice [16] .Yao et al., found that rhubarb supplementation increased the abundance of intestinal lactobacilli and bi dobacteria, and decreased the amount of intestinal Escherichia coli. In rats with severe acute pancreatitis [11] . These ndings are consistent with our study. Sennoside A exerted its corrective effects at phylum and genus levels, especially affecting the abundance of Faecalibacterium and Alistipes, which play key roles in brain function. Numerous studies showed that FMT is an effective method to modulate the dysbiosis of intestinal microbiota in many diseases [5,17] . This study showed that Sennoside A and FMT have similar effects in modulating the dysbiosis of intestinal ora in septic rats.
Previous human studies investigated the in uence of the intestinal microbiota on the brain and behavior, which is referred to as the gut-brain axis. Han et al., demonstrated decreased β diversity and intestinal microbiota richness (including Faecalibaculum) that led to cognitive dysfunction after surgery in APP/PS1 mice; however, probiotics' alteration of the intestinal microbiota attenuated this cognitive dysfunction [18] .
Alterations in gut microbial composition have also been observed in children with autism spectrum disorder (ASD), with a decrease in the Firmicutes/Bacteroidetes ratio [19] compared to children without ASD. Depression was negatively associated with Lachnospiraceae abundance. Patients with anxiety were characterized by elevated Bacteroidaceae [20] . Rifaximin administration is associated with microbial changes, including an increase in Eubacteriaceae and a decrease in Veillonellaceae abundance, which is accompanied by improved cognitive function in minimal hepatic encephalopathy [21] . Nogay et al., demonstrated that high growth rates of Clostridium histolyticum, C. perfringens, and Sutterella, high ratios of Escherichia/Shigella, and low ratios of Bacteroidetes/Firmicutes were generally related to GI problems, while relative abundance of Desulfovibrio, Clostridium spp., and Bacteroides vulgatus were associated with behavioral disorders. However, the available information is not yet detailed enough to develop a gut microbiota-based nutritional intervention to treat GI symptoms in people with autism [22] . Alistipes was overrepresented in patients with depression, an increase in Alistipes was observed in chronic social stress and was correlated with increases in proin ammatory cytokines IL-6 and TNF-α [23] . Notably, enrichment of Alistipes and reduction of Faecalibacterium, was shown to be associated with brain injury [24] . The genus Faecalibacterium is populated by bacterial species that produce SCFAs, which have an anti-in ammatory effect and are therefore bene cial to the host. Butyrate-producing Faecalibacterium and Coprococcus bacteria were consistently associated with higher quality brain function, and are implicated in relieving alcohol-induced anxiety [25] . This study proved that Sennoside A supplementation can correct the dysbiosis of intestinal ora structure in sepsis, especially the abundance of bacteria closely related to brain function, which may be a key reason why treatment with Sennoside A played an effective role in alleviating SAE symptoms.
The main pathogenesis of SAE is in ammation in the brain, including increased levels of TNF-α, IL-1β, and IL-6 in the cortex. Abnormal EEG can be used to diagnose SAE [26] . Evaluation of microbiota diversity indicated that a reduced microbial diversity is associated with in ammation, and changes in the relative abundance of Firmicutes and Bacteroidetes have been determined to affect the balance of in ammation. In particular, a rise in the Firmicutes/Bacteroidetes ratio is related to low-grade in ammation [27] . Reductions in the abundance of certain microbes, such as Faecalibacterium, which produce SCFAs, were associated with higher levels of in ammation in inflammatory bowel disease and autoimmune diseases [28] . Moreover, Faecalibacterium produces a 15kDa anti-inflammatory protein that inhibits the NF-κB pathway in intestinal epithelial cells and was shown to decrease the level NF-кB,IL-1β, IL-6 and TNF-α in a mouse model [29] . In addition, a rise in Alistipes has previously been observed in chronic social stress, and is correlated with increases in proin ammatory cytokines IL-6 and TNF-α [30] . This study showed that Sennoside A supplementation decreased the levels of IL-6, TNF-α, and IL-1β in SD rats' cortexes. In germ-free rats that received LPS, Sennoside A supplementation did not lower the ratios of abnormal EEGs, nor lower TNF-α, IL-1β, and IL-6 levels in rats'cortex. Sennoside A likely exerted its brain protective effects through gut microbiota alteration.

Conclusions
In summary, the present study demonstrated that Sennoside A supplementation and FMT can improve the diversity of SD rats' intestinal microbiota, and promotion of "bene cial bacteria" improved brain function. In short, Sennoside A can be used as a microbial regulator to improve LPS-associated encephalopathy. Sennoside A likely exerted its brain protective effects through gut microbiota alteration.

Declarations
This study was supported by the grants from Hebei science and technology planning project, China to Dr.

Availability of data and materials
The datasets used and/or analyzed in the current study are available from the corresponding authors on reasonable request.
Ethics approval and consent to participate All animal experimental protocols were approved by the Ethics Committee of Hebei General Hospital, and all procedures were performed in accordance with the Guideline for the Care and Use of Laboratory Animals from the National Institutes of Health, USA.

Consent for publication
Not applicable.

Competing interests
The authors declare no con ict of interest. All the authors listed have approved the manuscript.