Wolfberry, Yam, and Chrysanthemum polysaccharides increased intestinal Akkermansia muciniphila abundance and hepatic YAP1 expression to alleviate DILI

Drug‐induced liver injury (DILI) is frequently induced by high dose of acetaminophen (APAP) and is concomitant with disturbances of gut flora. Akkermansia muciniphila is beneficial for the repair of liver injury. Lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide all have anti‐inflammatory and antioxidation effects. The objective of this study is to investigate the potential of lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide (LYC) in improving DILI by increasing the abundance of A. muciniphila. Initially, screening for the optimal concentrations of wolfberry, yam, and chrysanthemum (WYC) or LYC to promote A. muciniphila proliferation in vitro and validated in antibiotic (ATB)‐treated KM mice. Subsequently, APAP‐induced DILI model in BALB/c mice were constructed to examine the treatment effects of LYC. Our findings indicate that the optimal concentration ratio of WYC was 2:3:2, and LYC was 1:1:1. WYC increased A. muciniphila proliferation in vitro and in ATB‐treated mice under this ratio. Meanwhile, LYC increased A. muciniphila abundance in vitro and the combination LYC with A. muciniphila promoted the proliferation of A. muciniphila in ATB‐treated mice. The overdose of APAP resulted in the impairment of the intestinal barrier function and subsequent leakage of lipopolysaccharide (LPS). Moreover, LYC increased A. muciniphila abundance, reduced intestinal inflammation and permeability, and upregulated the expression of the tight junction protein zonula occludens protein 1 (ZO‐1) and occludin contents in the gut. Lastly, LYC inhibited LPS leakage and upregulated hepatic YAP1 expression, ultimately leading to the repair of DILI.

vestigate the potential of lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide (LYC) in improving DILI by increasing the abundance of A. muciniphila.Initially, screening for the optimal concentrations of wolfberry, yam, and chrysanthemum (WYC) or LYC to promote A. muciniphila proliferation in vitro and validated in antibiotic (ATB)-treated KM mice.Subsequently, APAP-induced DILI model in BALB/c mice were constructed to examine the treatment effects of LYC.Our findings indicate that the optimal concentration ratio of WYC was 2:3:2, and LYC was 1:1:1.WYC increased A. muciniphila proliferation in vitro and in ATB-treated mice under this ratio.Meanwhile, LYC increased A. muciniphila abundance in vitro and the combination LYC with A. muciniphila promoted the proliferation of A. muciniphila in ATB-treated mice.The overdose of APAP resulted in the impairment of the intestinal barrier function and subsequent leakage of lipopolysaccharide (LPS).Moreover, LYC increased A. muciniphila abundance, reduced intestinal inflammation and permeability, and upregulated the expression of the tight junction protein zonula occludens protein 1 (ZO-1) and occludin contents in the gut.Lastly, LYC inhibited LPS leakage and upregulated hepatic YAP1 expression, ultimately leading to the repair of DILI.

| INTRODUCTION
Drug-induced liver injury (DILI) is frequently induced by large quantities acetaminophen (APAP) 1 and currently lacks effective treatment options. 2 Accumulating evidence has pointed out that hepatic injury is accompanied by an imbalanced gut flora, 3 and maintaining the integrity of the intestinal barrier promotes restore liver damage. 4oreover, oral administration of Akkermansia muciniphila enhanced colon mucosal thickness and inhibited hepatic inflammation in a liver injury mouse model. 5,6onsequently, it is urgent to investigate the changes in intestinal barrier and A. muciniphila abundance in the course of DILI and discover new antidotes.
8][9] Our previous studies have confirmed that APAP-induced DILI decreased hepatocellular YAP1 expression. 10Low expression level of YAP1 exacerbated liver damage in mice. 11Conversely, the high expression level of hepatic YAP1 was conducive to the repair of injured liver and intestine. 12,13Therefore, YAP1 in the liver is crucial to DILI recovery in mice.
Wolfberry, yam, and chrysanthemum all have protective effects on the liver in traditional Chinese medicine.5][16] Whether wolfberry, yam, and chrysanthemum (WYC) can repair DILI and enhance the intestinal barrier effect by increasing the abundance of A. muciniphila is unclear.Additionally, the treatment effects of the polysaccharides of lycium barbarum, yam, and chrysanthemum (LYC) relative to WYC are unknown and thus deserve in-depth study.Our study showed that LYC increased intestinal A. muciniphila abundance and barrier integrity, reduced LPS content levels, upregulated YAP1 expression in the liver, and alleviated DILI induced by APAP.

| Culture of A. muciniphila
The strain of A. muciniphila was purchased from ATCC (BAA-835).The culture methods were conducted according to our previous articles. 17The concentration of suspended A. muciniphila was 1 × 10 8 CFU/mL after 48 h of anaerobic culture.

A. muciniphila in vitro
Wolfberry, yam, and chrysanthemum were procured in Anguo.The polysaccharides of lycium barbarum, yam, and chrysanthemum (purity >90%) were acquired from Sichuan weikeqi Biotechnology.Three different concentrations were used and labeled low (L), medium (M), and high (H) according to Chinese pharmacopeia.The solution of wolfberry (L: 0.012 g/mL, M: 0.018 g/mL, and H: 0.024 g/mL), yam (L: 0.03 g/mL, M: 0.045 g/mL and H: 0.06 g/mL) or chrysanthemum (L: 0.01 g/mL, M: 0.015 g/ mL and H: 0.02 g/mL) were used for A. muciniphila intervention.][20] The culture medium was replaced with the drug-containing medium during A. muciniphila amplification.The optimal ratio of WYC and LYC to promote A. muciniphila abundance was determined via the orthogonal method.

| Animals
All animals were purchased from Beijing Vitonglihua Experimental Animal Technology and maintained under SPF conditions.This experiment was approved by the Animal Experiment Ethics Committee of Shanxi University of Chinese Medicine (AWE202305321) and Hebei University of Chinese Medicine (DWLL2021094).

| Preparation of WYC and LYC
According to the optimal ratio acquired by in vitro screening, the optimal concentration ratio of wolfberry: yam: chrysanthemum was determined to be 2:3:2 (Tables S1 and  S2), and the mixture was boiled in distilled water.The optimal concentration ratio of lycium barbarum polysaccharide: yam polysaccharide: chrysanthemum polysaccharide was 1:1:1 (Tables S3 and S4) and dissolved in normal saline.

K E Y W O R D S
Akkermansia muciniphila, drug-induced liver injury, intestinal barrier function, Lycium barbarum, the polysaccharides of lycium barbarum, yam and chrysanthemum, YAP1 | 3 of 15 LU et al.

| Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
The levels of ALT and AST were measured by kits (NJJC-BIO).The samples were measured at 510 nm with a microplate reader (Varioskan LUX, Thermo Fisher).Put the OD 510 value into the corresponding formula for calculation.

| Hematoxylin and eosin (H&E) staining
Hepatic and colonic tissues of mice were prepared by an automatic rotary microtome (RM2265).Next, staining with kit followed instruction (Beso).Pathological alterations were visualized under a microscope (DM2500).

| Immunohistochemistry
After antigen repair, hepatic tissue sections were stained using an immunohistochemical kit (ZSGB-BIO).The primary antibody was YAP (D8H1X) XP rabbit monoclonal antibody (14074S, CST, 1:200) and at 4°C overnight.DAB dye solution was then added according to the DAB kit (ZSGB-BIO).The expression of YAP1 in the hepatic nuclei of mice was observed and photographed under a microscope (DM2500).

| Quantitative real-time PCR
Fecal DNA extraction kit was used to extract fecal DNA (Solaibao).The amplification reactions contained DNA, primer, ddH 2 O, and Super Real PreMix Plus (Tiangen).Quantitative real-time PCR experiment was performed using Bio-Rad fluorescence quantitative PCR (Bio-Rad).Then, the data were processed and analyzed according to CT value and by the 2 −ΔΔCT method.The primer sequences of total bacteria and A. muciniphila were used according to our previous articles. 17

| 16S rRNA gene sequencing
Mice feces were collected and dispatched to Beijing Novogene Technology, and NovaSeq 6000 was used to detect bacterial diversity (V3-V4 region).The primer sequence was (F: CCTAY GGG RBG CAS CAG, R: GGACT ACN NGG GTA TCTAAT).The valuable data was obtained by splicing, filtering, and noise reduction of the original data.The specific composition of each sample was displayed at different species' taxonomic levels.

| Statistical analysis
SPSS 23.0 was used for statistical analyses in this study.Measurement data were expressed as mean ± SD.Measurement data following a normal distribution were compared using the t test.Multiple groups of measurement data were analyzed by one-way anova and Tukey test.The orthogonal experiment used three-way anova of variance and Range analysis.p < .05 was considered statistically significant.

| Wolfberry, yam, and chrysanthemum or lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide promoted the proliferation of A. muciniphila in vitro
It was found that increasing A. muciniphila abundance is advantageous for the repair of DILI in our previous research. 10Subsequently, we cultured the colony number of A. muciniphila with low, medium, and high doses of wolfberry or yam, or chrysanthemum in vitro (Figure 1A).The results confirmed that wolfberry, yam or chrysanthemum individually increased the abundance of A. muciniphila abundance in vitro (Figure 1B).
However, their synergistic effect in promoting A. muciniphila abundance remains unknown.An orthogonal table comprising three factors (wolfberry, yam, and chrysanthemum) and three levels (L, M, H) was designed to identify the optimal concentration ratio of WYC (Figure 1C).The result presented that the optimal concentration ratio was 2:3:2 (Tables S1 and S2), and it increased A. muciniphila abundance by 3.50-fold (Figure 1D).Notably, WYC outperformed wolfberry, yam, or chrysanthemum alone in vitro in terms of promoting the proliferation of A. muciniphila.
Lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide are the active ingredients of wolfberry, yam, and chrysanthemum, respectively.Nonetheless, their net effect in promoting the proliferation of A. muciniphila remains unclear.The orthogonal method results demonstrated that the optimal concentration ratio of LYC was 1:1:1 (Tables S3 and S4), and A. muciniphila abundance was increased by 3.76-fold (Figure 1F).Taken together, these results indicated that LYC was the principal component promoting the proliferation of A. muciniphila in WYC.

A. muciniphila promoted the abundance of A. muciniphila in ATB-treated mice
ATB water 10 was used to establish pseudointestinal germ-free mice models to explore the promoting effect of WYC or LYC on the abundance of A. muciniphila in vivo (Figure 2A).Intestinal total bacteria abundance was decreased, and A. muciniphila abundance was significantly diminished following ATB interference (Figure 2B,C), signifying that ATB exerted a vital effect on A. muciniphila abundance in the intestine of mice.Further, we found that the organ coefficient or body weight was unaltered after the administration of 1 × 10 8 CFU A. muciniphila (Figure 2D).At the same time, H&E staining revealed that the heart, liver, spleen, lung, kidney, and intestine of AKK and ATB mice exhibited a normal morphology without pathological phenomena compared with normal mice (Figure 2E).According to these results, 1 × 10 8 CFU A. muciniphila was not noxious to the organs in mice.
High A. muciniphila abundance enhanced the intestinal barrier effect of mice. 5To clarify its specific mechanism, we measured the contents of tight junction proteins ZO-1 and occludin, 22 intestinal inflammatory factors fecal zonulin and fecal calprotectin, 23 and gut permeability factor fecal α-1-antitrypsin. 24AKK increased ZO-1 content by 1.2-fold, while the content of occludin was unchanged (Figure 2F), implying that A. muciniphila enhanced tight junctions in the colon.Secondly, AKK adjusted the content of fecal zonulin and fecal α-1-antitrypsin to within normal ranges (Figure 2G,H).Collectively, these results insinuated that A. muciniphila enhanced the gut barrier by increasing tight junction proteins and limiting intestinal permeability and inflammation.
ATB decreased intestinal A. muciniphila abundance in mice.Besides, ATB increased the content of IL-6, a proinflammatory cytokine, 25 by 1.75-fold and that of LBP, a marker of intestinal microbial leakage, 26   The plating chart and counting histogram following A. muciniphila intervention in a medium containing wolfberry (L: 0.012 g/mL, M: 0.018 g/mL and H: 0.024 g/mL), yam (L: 0.03 g/mL, M: 0.045 g/mL and H: 0.06 g/ mL) or chrysanthemum (L: 0.01 g/mL, M: 0.015 g/mL, and H: 0.02 g/mL).(C) The orthogonal method was applied to explore the optimal concentration ratio of WYC to enhance A. muciniphila proliferation.(D) Medium containing with the optimal concentration ratio of WYC and the intervention of A. muciniphila in vitro.(E) The orthogonal method was used to explore the optimal concentration ratio of LYC to enhance A. muciniphila proliferation.(F) The optimal concentration ratio of LYC and the intervention of A. muciniphila in vitro.*p < .05.
the blood when the intestinal barrier is damaged. 27In addition, AKK inhibited the aberrant elevation in IL-6, LBP, and LPS levels induced by ATB in serum and readjusted these levels to normal range (Figure 2I,J).In conclusion, AKK intervention improved intestinal barrier integrity and inhibited LPS entry into the serum.
Unlike the in vitro results, A. muciniphila abundance was increased by 1.20-fold in WYC mice but reduced to 0.40-fold in WYC+AKK mice, decreased by 0.19-fold in LYC mice but increased by 1.66-fold in LYC+AKK mice, compared with normal mice (Figure 2K).These results conjointly indicated that WYC or LYC+AKK effectively increased intestinal A. muciniphila abundance of ATBtreated mice.

A. muciniphila in the gut of APAP-induced DILI mice
300 mg/kg APAP was used to construct DILI mice models (Figure 3A). 10 There was no significant difference in the total bacterial abundance, but A. muciniphila abundance was decreased to 0.08-fold after APAP intraperitoneal injection (Figure 3B).This result established a significant reduction in the abundance of intestinal A. muciniphila in following DILI.Furthermore, we uncovered that ATB reduced the overall bacterial abundance and community diversity in the colon of DILI mice, with A. muciniphila being the most affected (Figure 3C,D,F).
Akkermansia muciniphila abundance did not increase in WYC mice, was dramatically depleted in WYC+ATB mice, but increased by 2.54-fold in WYC+AKK mice compared with APAP mice.Moreover, WYC+AKK was more effective in promoting the abundance of A. muciniphila compared to AKK or NAC (Figure 3C).These findings inferred that WYC+AKK promoted intestinal A. muciniphila abundance of DILI mice.
We found that A. muciniphila was a biomarker in the gut of LYC mice through the species composition heat map (Figure 3H).LYC increased A. muciniphila abundance to 5.58-fold in the gut of DILI mice, which was superior to AKK and NAC.However, LYC+AKK intervention did not increase A. muciniphila abundance.Furthermore, the abundance of A. muciniphila was higher in LYC mice than in WYC+AKK mice (Figures 3C,E-G).These results suggested that LYC is more effective than WYC+AKK in promoting the abundance of A. muciniphila in the gut of DILI mice.

| lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide alleviated APAP-induced liver injury in BALB/c mice
Aspartate aminotransferase and alanine aminotransferase enzyme activity was increased by about 1.50-fold in APAP mice compared with normal mice, and punctate necrosis and infiltrated inflammatory factors could be observed in the liver of APAP mice (Figure 4A-C).This result validated that the model was successfully constructed.Interestingly, the content of IL-6, LBP, and LPS in the serum and liver of APAP mice were found to be increased by approximately 2.00-and 2.50-fold, respectively, compared to normal mice (Figure 4D-F1).These results indicated that a higher concentration of LPS triggered the synthesis of proinflammatory factors following DILI.Furthermore, the intervention of ATB exacerbated hepatic damage, as evidenced by a roughly 2.5-fold increase in AST and ALT enzyme activity compared to normal mice.Additionally, inflammatory factors were observed in the liver of APAP+ATB mice (Figure 4A-C).
Earlier studies have documented that high A. muciniphila abundance improved the status quo of DILI. 10 LYC markedly increased A. muciniphila abundance in this study.Furthermore, LYC adjusted AST and ALT enzyme activity to normal levels and reduced IL-6, LPS, and LBP contents to approximately 0.70-fold in the liver of APAP mice.Besides, the liver had no pathological changes were identified in the liver of LYC mice (Figure 4A-C,D1-F1).The degree of liver recovery in LYC mice was similar to those in the NAC and AKK groups (Figure 4A-C), indicating that LYC facilitated APAP-induced DILI repair.
As mentioned earlier, the abundance of A. muciniphila increased in WYC+AKK mice but decreased in WYC+ATB mice.Besides, both WYC and WYC+AKK exerted excellent effects on the damaged liver, as evidenced by the normalization of AST and ALT enzyme activity and the restoration of normal liver structure (Figure 4A-C).Likewise, the contents of IL-6, LPS, and LBP were also decreased in the liver of WYC+AKK mice (Figure 4D-F1).However, WYC+ATB increased AST and ALT enzyme activity.Besides, the liver structure was altered, and inflammatory factors were detected in the liver of WYC+ATB mice (Figure 4A-C), suggesting that WYC did not promote liver repair in the presence of an imbalanced flora.

| lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide enhanced the intestinal barrier effect of APAP-induced DILI mice
Lipopolysaccharide leakage was noted in APAP mice of this study, which is associated with impaired intestinal barrier function. 27Besides, the distribution of epithelial cells was found to be uneven, accompanied by increased levels of inflammatory factors and a decreased number of goblet cells, which relieves intestinal inflammation, 28 in the gut of APAP mice (Figure 5A,B).Contrastingly, the contents of fecal calprotectin, fecal zonulin, and fecal α-1-antitrypsin were increased by 2.50-, 1.43-and 3.54-fold, respectively, in APAP mice compared with normal mice (Figure 5C-E).Afterward, we found that the contents of ZO-1 and occludin were decreased to nearly 0.50-fold in APAP mice compared with normal mice (Figure 5F,G).In short, high doses of APAP resulted in an increase in intestinal inflammatory factors and permeability and destroyed tight junctions, ultimately leading to LPS release.
Additionally, this study identified the presence of a congestive and edematous basal layer was noted in the intestine of APAP+ATB mice, as well as an increased proportion of foreign bodies (Figure 5A).Furthermore, fecal calprotectin content was increased by 2.96-fold in the gut of APAP+ATB mice compared with normal mice (Figure 5C).These results established that the imbalance of intestinal flora impaired the gut barrier.
In addition to mitigating liver damage, we discovered that LYC mice exhibited well-organized epithelial cells and an augmented presence of goblet cells in the colon (Figure 5A,B).Notably, LYC also decreased fecal calprotectin and fecal α-1-antitrypsin contents to about 0.70-fold, and increased ZO-1 and occludin contents by about 2.00fold compared with APAP mice (Figure 5C-G).In short, LYC exerted advantageous effects in limiting intestinal inflammation and enhancing tight junctions compared with AKK or NAC, demonstrating its protective effects against gut damage in DILI mice.Consistent with the aforementioned results, WYC played a beneficial role in APAPinduced gut damage when combined with A. muciniphila but did not have an excellent effect when gut flora was imbalanced (Figure 5A-G).It is worthwhile emphasizing that a higher proportion of goblet cells was present in LYC mice than in WYC+AKK mice (Figure 5B), resulting in robust tight junctions in the gut.To sum up, LYC exerted superior efficacy in promoting intestinal tight junctions compared to WYC.

| lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide upregulated hepatic YAP1 expression in APAP-induced DILI mice
Prior studies evinced that hepatocellular YAP1 expression was downregulated in APAP-induced DILI mice. 10 In the current study, the expression level of YAP1 in the hepatic nucleus was found to be lower in APAP+ATB mice compared with APAP mice (Figure 6C,I), indicating that ATB inhibited the upregulation of YAP1 expression in the hepatic nuclei.
More importantly, the expression level of YAP1 was increased by roughly 2.50-fold in the liver of LYC mice, higher than that of AKK or NAC mice (Figure 6H,I).However, there was no significant difference in YAP1 expression between LYC+AKK mice and APAP mice (Figure 6F,I).These results corroborated that LYC effectively enhanced YAP1 expression in the liver.Furthermore, the YAP1 expression level was increased by about 1.50-fold in WYC mice and 3.50-fold in WYC+AKK mice but was not significantly different in WYC+ATB mice compared with APAP mice (Figure 6G,I).This result signaled that WYC improved hepatic YAP1 expression through A. muciniphila.However, WYC+AKK increased YAP1 expression by about 1.40-fold compared with LYC mice, implying that WYC+AKK was the most effective in promoting hepatic YAP1 expression (Figure 6H,I).

| DISCUSSION
Drug-induced liver injury is a hepatic disease with limited treatment strategies and is typically caused by high LYC showed promise as a potential treatment of APAPinduced DILI.
Despite decades of study, the mechanism of APAPinduced DILI is not fully understood.In addition to causing liver damage, excessive APAP also resulted in transient increased intestinal permeability and intestinal apoptosis. 29This study confirmed for the first time APAP damaged intestine and downregulated the gut barrier function.We found that high dose of APAP activated the inflammatory response, increased permeability, and weakened tight junctions in the gut.The damaged intestinal barrier caused LPS extravasation into the liver. 30ignificantly, we observed a higher concentration of LPS in the liver compared to the serum.This elevated LPS concentration subsequently triggered the synthesis of proinflammatory factors, such as IL-6, in the liver following DILI.These findings indicated that overdose APAP activated intestinal inflammatory response and destroyed Besides, LPS downregulated YAP1 expression and accelerated the death of cardiomyocytes. 31To date, the relationship between LPS and YAP1 in hepatocytes remains underexplored.Our study further elucidated that YAP1 expression was downregulated in the liver of mice with elevated LPS levels, indicating a potential correlation between heightened hepatic LPS concentrations and diminished YAP1 expression in hepatocytes.
Except for the integrity of intestinal barrier, the homeostasis of gut microbes also plays a critical role in promoting the repair of liver disease. 27However, the precise role of gut microbiota in APAP-induced DILI remains unclear.Ampicillin disrupts intestinal microecology, significantly decreases Lactobacillus abundance, and aggravates hepatic injury in APAP-induced acute liver failure. 32Clinical case reports described that A. muciniphila abundance was reduced in patients with alcoholic fatty liver. 33Similarly, this study determined that the intestinal A. muciniphila abundance was markedly decreased after being intraperitoneally injected with a high dose of APAP.Herein, the microbial diversity was decreased in ATB-treated DILI mice, particularly with a significant decrease in the abundance of A. muciniphila.Besides, the severity of intestinal lesions was heightened, causing bacterial translocation and inflammatory responses in the liver, thereby exacerbating hepatic injury.A previous study evinced that the imbalance of intestinal flora was negatively correlated with the degree of DILI, 34 which was consistent with the findings of this study.
Akkermansia muciniphila is recognized as a marker of intestinal microbes in healthy individuals, 35 it is beneficial for reducing hepatic inflammatory. 6Other than that, clinical studies reported that 1 × 10 10 CFU A. muciniphila diminished the content of AST, ALT, and LPS in obese human serum, 36 but the underlying mechanism has not been elucidated so far.We demonstrated that 1 × 10 8 CFU A. muciniphila did not cause organ toxicity in ATB-treated mice.Meanwhile, it increased the integrity of tight junctions and decreased inflammation and permeability in the gut.An intact intestinal barrier and high A. muciniphila abundance are favorable for repairing liver injury. 33The enhancement of the intestinal barrier inhibited LPS release, which is beneficial to the repair of liver injury, 37 as demonstrated in this study.Taken together, taking a safe dose of A. muciniphila has the effect of promoting intestinal barrier integrity.
Currently, there are few studies of improving DILI with traditional Chinese medicine.9][40] Interestingly, our study found that WYC upregulated the expression of hepatic YAP1 and mitigated DILI caused by APAP, despite not elevating A. muciniphila abundance.This result signaled that WYC has the potential to enhance hepatic YAP1 expression and assist in the repair of liver injury.More interestingly, the WYC+AKK mice exhibited a notable enhancement in A. muciniphila abundance and tight junction, along with a reduced inflammatory response and permeability in the gut.Additionally, there was an increase in hepatic YAP1 expression in these mice.In comparison, the WYC+ATB mice decreased intestinal barrier function and hepatic YAP1 expression, accompanied by aggravated liver damage.Based on these findings, we speculate that WYC promoted hepatic YAP1 expression through A. muciniphila, and the high abundance of A. muciniphila was conducive to the involvement of WYC in the repair of liver injury.
Among three polysaccharides, only lycium barbarum polysaccharide has been reported to participate in liver repair. 41And this study found that LYC markedly increased intestinal A. muciniphila abundance.Besides, LYC significantly increased the number of goblet cells, indicating a reinforcement of the intestinal barrier. 42This reinforcement was accompanied by an enhancement of tight junctions, a reduction in inflammation and permeability within the gut.Consequently, the enhanced gut barrier subsequently inhibited LPS leakage and reduced the levels of proinflammatory factors in the liver.Furthermore, LYC upregulated hepatic YAP1 expression, thereby facilitating the repair of liver injury.Therefore, we postulate that LYC exhibits superior efficacy compared to WYC alone in repairing APAP-related hepatic and intestinal injury.
In short, high doses of APAP resulted in DILI, disruption of the intestinal barrier, promoted LPS leakage into the liver, and downregulated the expression of hepatic YAP1. A. muciniphila enhanced the gut barrier function and mitigated DILI damage.Thereafter, LYC increased the abundance of A. muciniphila and the number of goblet cells, strengthened tight junctions, and minimized intestinal inflammation and permeability.As a result, the enhanced intestinal barrier subsequently inhibited LPS leakage, reduced the contents of hepatic proinflammatory factors in the liver and upregulated hepatic YAP1 expression, finally aiding DILI repair.Consequently, LYC possesses promising treatment value in repairing APAPassociated DILI.

F I G U R E 1
Wolfberry (WYC) or lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide (LYC) promoted the proliferation of Akkermansia muciniphila in vitro.(A) The schematic diagram of cultured A. muciniphila in a medium containing wolfberry, yam, or chrysanthemum in vitro.(B)

F I G U R E 2
Wolfberry or the combination of lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide and Akkermansia muciniphila promoted the abundance of A. muciniphila in ATB-treated mice.(A) Model establishment schematic of pseudointestinal germ-free mice.(B) The abundance of total bacteria and A. muciniphila in normal and ATB mice was detected by realtime PCR after model construction.(C) Culture results of activated-A.muciniphila in feces of normal mice and ATB mice after model construction.(D) Weight changes and organ coefficients of Normal, ATB, and AKK mice.(E) The pathological images of H&E staining in heart, liver, spleen, lung, kidney and intestine sections (scale bar, 20 μm).(F) ZO-1 and occludin contents in the colon of mice.(G, H) Fecal calprotectin, fecal zonulin, and fecal α-1-antitrypsin contents in the feces of mice.(I, J) IL-6, LPS, and LBP contents in the serum of mice.(K) The abundance of total bacteria and A. muciniphila in the colon of each group of mice via real-time PCR.*p < .05. ns, no statistical significance.

F I G U R E 3
lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide increased the abundance of Akkermansia muciniphila in the gut of APAP-induced DILI mice.(A) Diagram of the APAP-induced DILI mouse model.(B, C) Fluctuations in the total bacteria and A. muciniphila of mice by real-time PCR after membrane formation or intervention.(D) Shannon index plot of 16S rRNA gene sequencing in each group of mice.(E) Composition of microflora (top 10) at the phylum level in each group of mice.(F) Composition of microflora (top 20) at the genus level in each group of mice.(G) Heat map displaying the top twenty species at the genus level in each group of mice.(H) Histogram linear discriminant analysis among each group of mice.*p < .05 and **p < .01. ns, no statistical significance.doses of APAP. 2 Much evidence point out that the increased abundance of A. muciniphila and enhanced gut barrier have positive effects on the repair of liver injury. 4,10Therefore, we mainly focus the effect of LYC on A. muciniphila abundance and the integrity of the gut barrier in DILI mice.This study revealed that LYC markedly increased intestinal A. muciniphila abundance enhanced the intestinal barrier integrity, subsequently inhibited LPS leakage into the liver, elevated the expression of hepatic YAP1, and alleviated APAP-induced DILI.Therefore, F I G U R E 4 lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide alleviated APAP-induced liver injury in BALB/c mice.(A, B) AST and ALT hepatic enzyme activity in each group of mice.(C) The hepatocyte morphology of liver sections stained by H&E (scale bar, 20 μm, a representing inflammatory factors infiltrating the portal area, b delineating necrosis of punctate foci, c displaying hepatocyte lysis necrosis).(D-F1) IL-6, LPS, and LBP contents in the serum and liver.*p < .05.

F I G U R E 5
lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide enhanced the intestinal barrier effect of APAP-induced DILI mice.(A) The pathological morphology of the colon by H&E staining (scale bar, 20 μm, a illustrating the uneven arrangement of epithelial cells in the mucosal layer of the colon, b exhibiting infiltration of inflammatory cells in the mucosal layer of the colon, c indicating a decrease in the number of goblet cells, d indicating hyperemia and edema in the basal layer of the colon, e showing the loss of colonic serosa and deposition of foreign bodies, f representing abnormal villi arrangement, g showing the disrupted intestinal gland structure).(B) PAS staining of the colon (scale bar, 20 μm, goblet cells were purplish red).(C-E) Fecal calprotectin, fecal zonulin, and fecal α-1-antitrypsin contents in feces.(F, G) ZO-1 and occludin contents in the colon.*p < .05.

F I G U R E 6
lycium barbarum polysaccharide, yam polysaccharide, and chrysanthemum polysaccharide upregulated hepatic YAP1 expression in APAP-induced DILI mice.(A-H) Hepatic YAP1 expression of DILI mice as determined by western blot.GAPDH was used as the internal reference.(I) The nuclear expression of YAP1 in DILI mice liver by immunohistochemistry staining (scale bar, 20 μm).*p < .05. | 13 of 15 LU et al. intestinal barrier function, allowing LPS to enter the liver and initiate hepatic inflammation.
by 1.60-fold, in the serum of mice.LPS, bound with LBP, is detected in