Histamine Causes Pyroptosis of Liver by Regulating Gut-Liver Axis in Mice

Huangjiu usually caused rapid-drunkenness and components such as β-benzyl ethanol (β-be), isopentanol (Iso), histamine (His), and phenethylamine (PEA) have been reported linked with intoxication. However, the destructive effect of these components on gut microbiota and liver is unclear. In this study, we found oral treatment of these components, especially His, stimulated the level of oxidative stress and inflammatory cytokines in liver and serum of mice. The gut microbiota community was changed and the level of lipopolysaccharide (LPS) increased significantly. Additionally, cellular pyroptosis pathway has been assessed and correlation analysis revealed a possible relationship between gut microbiota and liver pyroptosis. We speculated oral His treatment caused the reprogramming of gut microbiota metabolism, and increased LPS modulated the gut-liver interaction, resulting in liver pyroptosis, which might cause health risks. This study provided a theoretical basis for the effect of Huangjiu, facilitating the development of therapeutic and preventive strategies for related inflammatory disorders.


Introduction
The cultivation and use of fermented wines can be traced back to 7000 years ago. The typical production technology is via a solid or semi-solid brewing mode of 'bilatera fermentation' with single or multiple grains such as glutinous rice, sorghum, wheat, corn, and rice as raw materials and wheat koji as a saccharifying starter [1,2]. During the above open fermenting process, various strains of yeast, bacteria, fungi, mold and other strains inoculated naturally or cooperatively undergo glucose metabolism, protein metabolism, and lipid metabolism. At the same time, complex flavor compounds were produced [2]. A variety of fermented wines have been developed and consumed in Asian countries. Recently, fermented foods are of public interest and consumed more frequently in western countries because fermented foods, particularly fermented wine, have been proven to have health-promoting and protective effects.
Huangjiu is a nutritive brewing wine made from rice, millet, corn, and other grains through cooking, saccharification, fermentation, filtration, and frying [3], thus Huangjiu is a complex mixture composed of many chemical components besides ethanol [4]. Carbohydrates, amino acids, organic acids, lipids are the main functional components in Huangjiu [3]. However, there are compounds in Huangjiu that may adversely affect its flavor, taste, and even safety of it [5]. Rapid-drunkenness has always been one of the most important factors affecting the consumers' choice of Huangjiu, which has seriously limited the development of the Huangjiu industry. It has been reported that compounds such as The results for the body weight, liver weight, serum, and liver biomarkers, such as MDA, GSH, SOD were presented in Figure 1A-I. There was no significance in body weight among groups ( Figure 1A). Although the liver weight of β-be, His, Iso, and PEA groups was significantly decreased when compared with Normal (p < 0.01, Figure 1B), there was no significance between the ratios of liver weight to body weight ( Figure 1C). The serum GSH, SOD, MDA of the Iso group were significantly higher than other groups (p < 0.01, Figure 1D-F). The liver GSH, SOD, MDA of His group were significantly lower than any other group (p < 0.01, Figure 1G-I). The serum GSH, SOD, MDA of the Iso group were significantly higher than other groups (p < 0.01, Figure 1D-F). The liver GSH, SOD, MDA of His group were significantly lower than any other group (p < 0.01, Figure 1G-I).

Effects of His, PEA, β-Be, and Iso on Inflammatory Cytokines in the Liver and Serum
H&E staining has been performed to assess the effect of different components on the liver. As shown in Figure 2A-F, among the five treated groups, the His group exhibited the most significant difference when compared to the Normal group. Hyperchromatic nuclei and invisible nucleoli were present in the His group. Hepatocytes with two nuclei were common, and other hepatocytes were in the mitotic phase. Liver cells with pathological changes can also be seen in the His group, such as lytic cells, disappeared nucleus, white vacuoles in the cytoplasm, and vacuolated lesions. The lesions observed occurred in more areas in His group compared with other groups (Figure 2A-F). Meanwhile, the His groups showed higher significance in TNF-α and IL-10 levels both in the liver and serum (p < 0.01, Figure 2G,H). However, the level of IL-6 had no significant difference among groups ( Figure 2I). 2.2. Effects of His, PEA, β-Be, and Iso on Inflammatory Cytokines in the Liver and Serum H&E staining has been performed to assess the effect of different components on the liver. As shown in Figure 2A-F, among the five treated groups, the His group exhibited the most significant difference when compared to the Normal group. Hyperchromatic nuclei and invisible nucleoli were present in the His group. Hepatocytes with two nuclei were common, and other hepatocytes were in the mitotic phase. Liver cells with pathological changes can also be seen in the His group, such as lytic cells, disappeared nucleus, white vacuoles in the cytoplasm, and vacuolated lesions. The lesions observed occurred in more areas in His group compared with other groups (Figure 2A-F). Meanwhile, the His groups showed higher significance in TNF-α and IL-10 levels both in the liver and serum (p < 0.01, Figure 2G,H). However, the level of IL-6 had no significant difference among groups ( Figure 2I).

Effects of His, PEA, β-Be, and Iso on Pyroptosis in the Liver
As shown in Figure 3A-C, the level of Cas-1, GSDMD, and IL-1β in the His group were significantly enhanced compared with those in the other groups (p < 0.01). A similar tendency was seen with the level of Cas-1 and IL-1β in the Iso group. The level of gut LPS was presented in Figure 3D, and we found that the LPS level was significantly higher in the His group than in the other groups (p < 0.01). 2.3. Effects of His, PEA, β-Be, and Iso on Pyroptosis in the Liver As shown in Figure 3A-C, the level of Cas-1, GSDMD, and IL-1β in the His group were significantly enhanced compared with those in the other groups (p < 0.01). A similar tendency was seen with the level of Cas-1 and IL-1β in the Iso group. The level of gut LPS was presented in Figure 3D, and we found that the LPS level was significantly higher in the His group than in the other groups (p < 0.01).

Effects of His, PEA, β-Be, and Iso on Gut Microbial Community Structure and Composition
Total DNA was extracted from the feces samples and the 16S rRNA gene of V3-V4 was amplified by PCR. Sequencing of PCR products was performed by using the Illumina MiSeq platform at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). After removing the low-quality and chimeric sequences, 780,661 high-quality sequencing reads were generated from feces samples. A total of 613 operational taxonomic units (OTUs) were generated from the high-quality sequences at 97% sequence. Ace and Simpson indexes of the gut microbiome in the His group and the β-be group significantly decreased ( Figure 4A,D). Shannon index of the gut microbiome in the His group and the Eth group significantly decreased ( Figure 4C). However, there is no significance of the Chao index between the treated groups and the Normal group ( Figure 4B). LEfse analysis further confirmed the difference of gut microbial structures between all groups ( Figure 4E). At the phyla level, there were 19 phyla were identified. The dominant phylum with relative abundance > 1% was Bacteroides (72.75%), Firmicutes (25.09%), and Proteobacteria (1.39%) ( Figure 5A) in all groups. At the genus level, there were 213 genera were identified. The dominant genera were considered as norank_f__Muribaculaceae (32.47%), Prevotellaceae_UCG-001 (22.61%), Lachnospiraceae_NK4A136_group (3.24%), unclassified_f__Lachnospiraceae (6.54%), and Alistipes (4.86%) ( Figure 5B). We found that the His group decreased the relative abundance of norank_f__Muribaculaceae compared with that of the Normal group. The His, Eth, βbe, PEA group significantly increased the relative abundance of Prevotellaceae_UCG_001 (p < 0.01). The gut microbiome composition in His group mice differed substantially from those in the mice in Iso, Eth, and PEA group ( Figure

Effects of His, PEA, β-Be, and Iso on Gut Microbial Community Structure and Composition
Total DNA was extracted from the feces samples and the 16S rRNA gene of V3-V4 was amplified by PCR. Sequencing of PCR products was performed by using the Illumina MiSeq platform at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). After removing the low-quality and chimeric sequences, 780,661 high-quality sequencing reads were generated from feces samples. A total of 613 operational taxonomic units (OTUs) were generated from the high-quality sequences at 97% sequence. Ace and Simpson indexes of the gut microbiome in the His group and the β-be group significantly decreased ( Figure 4A,D). Shannon index of the gut microbiome in the His group and the Eth group significantly decreased ( Figure 4C). However, there is no significance of the Chao index between the treated groups and the Normal group ( Figure 4B). LEfse analysis further confirmed the difference of gut microbial structures between all groups ( Figure 4E). At the phyla level, there were 19 phyla were identified. The dominant phylum with relative abundance > 1% was Bacteroides (72.75%), Firmicutes (25.09%), and Proteobacteria (1.39%) ( Figure 5A) in all groups. At the genus level, there were 213 genera were identified. The dominant genera were considered as norank_f__Muribaculaceae (32.47%), Prevotellaceae_UCG-001 (22.61%), Lachnospiraceae_NK4A136_group (3.24%), unclassi-fied_f__Lachnospiraceae (6.54%), and Alistipes (4.86%) ( Figure 5B). We found that the His group decreased the relative abundance of norank_f__Muribaculaceae compared with

Correlation among Gut Microbiota, Liver Pyroptosis and Microbial Metabolism
In order to explore the potential relationship between gut and liver, we applied the correlation analysis among gut microbiota, gut LPS, and the levels of IL-1β, Cas-1, and GS-DMD through Spearman correlation analysis ( Figure 6). As shown in the figure, the relative abundance of Pseudogracbacilus negatively correlated with the level of Cas-1 (p < 0.05), but significant positively correlated with the level of GSDMD (p < 0.01); the relative abundance of Sporosarcina negatively correlated with the levels of gut LPS and IL-1β (p < 0.05); the relative abundance of norank_f_Lachnospiracae negatively correlated with the level of GSDMD but positively correlated with the level of Cas-1 and IL-1β (p < 0.05; p < 0.01, respectively). We also found that the relative abundance of Lachnospiraceae_NK4A136_group negatively correlated with the level of GSDMD (p < 0.05) and positively correlated with the level of Cas-1 (p < 0.05). in His group mice differed substantially from those in the mice in Iso, Eth, and PEA group ( Figure 5C,D).    in His group mice differed substantially from those in the mice in Iso, Eth, and PEA group ( Figure 5C,D).   < 0.05), but significant positively correlated with the level of GSDMD (p < 0.01); the relative abundance of Sporosarcina negatively correlated with the levels of gut LPS and IL-1β (p < 0.05); the relative abundance of norank_f_Lachnospiracae negatively correlated with the level of GSDMD but positively correlated with the level of Cas-1 and IL-1β (p < 0.05; p < 0.01, respectively). We also found that the relative abundance of Lachnospi-raceae_NK4A136_group negatively correlated with the level of GSDMD (p < 0.05) and positively correlated with the level of Cas-1 (p < 0.05).

Discussion
The mice model is one of the commonly used models to investigate liver damage and has long been used to evaluate the pathological process of alcoholic liver disease (ALD) [17], nonalcoholic liver disease (NALD) [11], and other liver injuries [10]. Compared with gavage, intraperitoneal injection greatly shortens the modeling cycle, but there is a higher risk of death concurrently [18]. In the present study, we investigated the effect of four main components in Huangjiu, His, PEA, β-be, and Iso, on liver pyroptosis by oral treatment in mice. We first investigated the effects of four components on body weight, liver weight, and level of oxidative stress in serum and liver of mice. Simultaneously, the level of inflammatory cytokines in the liver and serum were examined. Results have shown that treatment with four components, especially the His, significantly stimulated the level of oxidative stress and inflammatory cytokines both in serum and liver. In order to explore the underlying mechanism, we performed gut microbiota analysis and pyroptosis pathway assay and found the gut microbial community structure and composition has been

Discussion
The mice model is one of the commonly used models to investigate liver damage and has long been used to evaluate the pathological process of alcoholic liver disease (ALD) [17], nonalcoholic liver disease (NALD) [11], and other liver injuries [10]. Compared with gavage, intraperitoneal injection greatly shortens the modeling cycle, but there is a higher risk of death concurrently [18]. In the present study, we investigated the effect of four main components in Huangjiu, His, PEA, β-be, and Iso, on liver pyroptosis by oral treatment in mice. We first investigated the effects of four components on body weight, liver weight, and level of oxidative stress in serum and liver of mice. Simultaneously, the level of inflammatory cytokines in the liver and serum were examined. Results have shown that treatment with four components, especially the His, significantly stimulated the level of oxidative stress and inflammatory cytokines both in serum and liver. In order to explore the underlying mechanism, we performed gut microbiota analysis and pyroptosis pathway assay and found the gut microbial community structure and composition has been changed, resulting in the increase of inflammatory LPS and subsequent pyroptosis of liver cells. Correlation analysis indicated the possible relationship between oral treatment, gut microbiota metabolism, and liver pyroptosis.
Alcohol can induce the increase of endotoxin level, activate Kupffer cells, and trigger the excessive production of TNF-α, IL-1β, and IL-6, which participate in the pathogenesis of liver diseases [19]. TNF-α could induce SREBP1 expression, resulting in hepatic steatosis [20]; IL-1β and IL-6 are involved in hepatic steatosis and inflammation, which causes hepatic disease [21]. In this study, we found that besides ethanol, the expression of TNF-α, IL-1β, and IL-6 in other treated groups increased significantly, which might trigger liver inflammation, directly or indirectly.
Pyroptosis is a programmed cell death characterized by the formation of pores on the plasma membrane, cell swelling, and plasma membrane rupture, similar to necrosis rather than apoptosis [22]. For a long time, IL-1β has been considered an important pyrogen. IL-1β affects the body's innate and adaptive immunity through different mechanisms, so as to significantly enhance the body's response to infectious diseases [23]. Although pyroptosis in host defense is largely unclear, it has been proved to inhibit the replication and proliferation of microorganisms in vivo [24]. The proinflammatory effect of IL-1β and pyroptosis could induce autoimmune and inflammatory diseases [25]. As recently reported, Gasdermin D (GSDMD) protein was presented in nigericin-induced NLRP3 inflammasomes, which act on the prediction site of inflammatory activation and participate in pyroptosis and IL-1β secretion [14]. In the present study, the level of GSDMD and IL-1β of the liver in the His group both increased significantly, indicating that pyroptosis caused by His occurred in the liver.
Intestinal homeostasis is maintained by the interrelationship between the gut microbiota, intestinal barrier, and immune system. Imbalance in the gut microbiome often tends to result in the destruction of the intestinal barrier and immune system [26]. Under normal physiological conditions, intestinal epithelial cells, microbiota, and immune cells support the steady-state of the intestinal system collaboratively. Intestinal epithelial cells receive signals from the microbiota, such as microbial metabolites (LPS, short-chain fatty acids, etc.) or the microbes per se, so as to maintain the normal physiological function of the mucosal barrier [27]. LPS is associated with excessive inflammatory response, mainly activating NFκB by binding to peripheral blood macrophages or microglia TLR4/CD14 complex, increasing the production of cytokines such as IL-6 and TNF-α to cause an inflammatory response [28]. In this study, the level of the gut LPS was observed to increase significantly in the His group. Meanwhile, the level of liver IL-6 and TNF-α in the His group increased either, which means the increase of gut LPS might cause liver inflammation, connecting the gut microbiota change and the liver pyroptosis. Nevertheless, the gut microbiota could also regulate host immune function through the action of metabolites or endotoxin [29]. At the same time, immune cells can directly or indirectly affect the growth of the microbiota by releasing cytokines or chemokines [30].
To further explore the underlying mechanism, the effects of His, PEA, β-be, and Iso on the gut microbiota were assessed by 16S rRNA sequencing. We found that His treatment increased the relative abundance of Proteobacteria at the phylum level, which was considered as a microbial marker for dysbiosis [31]. At the genus level, His treatment enriched the relative abundance of Lachnospiraceae_NK4A136_group and norank_f _Lachnospiracae, which belong to the Lachnospiraceae family, and can also be enriched by the Fubrick tea in the previous study [32]. Through the correlation analysis among gut microbiota, LPS, and the levels of IL-1β, Cas-1 and GSDMD, we found that the relative abundance of Lach-nospiraceae_NK4A136_group negatively correlated with the level of GSDMD (p < 0.05) and positively correlated with the level of Cas-1 (p < 0.05). Lachnospiraceae_NK4A136_group was reported to negatively correlate with obesity [33]. In our study, the body weight of mice in His group was decreased, and we speculated that Lachnospiraceae_NK4A136_group caused pyroptosis and inflammation in the liver, resulting in weight loss.
In conclusion, the treatment of four main components in Huangjiu, especially the histamine, caused metabolic disorder, oxidative stress, liver pyroptosis, and inflammation via regulating gut microbiota and its metabolism. The possible mechanism was summarized and illuminated in Figure 7. To sum up, oral histamine treatment caused changes in the gut microbiota structure and composition, especially Lachnospiraceae_NK4A136_group and norank_f_norank_o_Clostridia_UGG-014, resulting in the increase of inflammatory LPS, which subsequently caused oxidative stress and inflammatory damage to the liver, further lead to liver pyroptosis and may even cause health risks. Although the present study analyzed the destructive effect of main components in Huangjiu on liver and investigated the possible underlying mechanism, there were still some possible limitations in this study. What is the exact source of gut LPS and how the main components in Huangjiu triggered its promotion? Is there any synergetic or antagonistic effect of the four main components? There are too many unresolved questions to be answered. Further studies focusing on the comprehensive effect study of combination of main components, the effective removal of harmful components, as well as other possible harmful effects study of Huangjiu exert on human health, are needed. Overall, the study revealed a possible relationship between gut microbiota and liver metabolism, and the obtained results might help guiding the establishment of a recommended limiting standard of harmful components in Huangjiu, which shed light on the gut-liver axis research and contribute to the development of Huangjiu industry, and should be further investigated.
which subsequently caused oxidative stress and inflammatory damage to the liver, further lead to liver pyroptosis and may even cause health risks. Although the present study analyzed the destructive effect of main components in Huangjiu on liver and investigated the possible underlying mechanism, there were still some possible limitations in this study. What is the exact source of gut LPS and how the main components in Huangjiu triggered its promotion? Is there any synergetic or antagonistic effect of the four main components? There are too many unresolved questions to be answered. Further studies focusing on the comprehensive effect study of combination of main components, the effective removal of harmful components, as well as other possible harmful effects study of Huangjiu exert on human health, are needed. Overall, the study revealed a possible relationship between gut microbiota and liver metabolism, and the obtained results might help guiding the establishment of a recommended limiting standard of harmful components in Huangjiu, which shed light on the gut-liver axis research and contribute to the development of Huangjiu industry, and should be further investigated.

Animals and Experimental Treatment
Thirty-six male C57BL/6J mice (6-8 weeks old, 25-30 g, Specific Pathogen Free) and fodder were obtained from Beijing Vital River Laboratory Animal Center [SCXK(Jing)2016-0006]. The mice were acclimated for 1 week before the experiments. The mice were housed in a room with a controlled temperature (22 ± 2 • C), humidity (65% ± 5%), and 12 h dark/light cycle. After the acclimation period, mice were randomly divided into six groups(n = 6): Normal (normal control: feed with normal saline,15 mL/kg BW), Eth (15% (v/v) ethanol solution, 15 mL/kg BW), His (histamine dissolved in 15% (v/v) ethanol solution, 15 mL/kg BW), PEA (phenethylamine prepared in 15% (v/v) ethanol solution, 15 mL/kg BW), β-be (β-benzyl ethanol dissolved in 15% (v/v) ethanol solution, 15 mL/kg BW), Iso (isopentanol prepared in 15% (v/v) ethanol solution, 15 mL/kg BW) ( Figure S1). The dosages of these substances above were set according to Peng et al. [34]. According to the weight of 60 kg adult drink 2 bottles of 500 mL Huangjiu, according to the concentration of each substance in Huangjiu, we calculated the content of substance intake per kg body weight of an adult, and the corresponding dose is determined by the intake per kg body weight of mice. The specific parameters were shown in Table S1. All groups received normal foods and water. The treatment was given by gavage once a day for 2 weeks; body weight was measured every other day.
After the experiment, the mice feces were collected in the sterile sampling tube and frozen at −80 • C for later use. At the end of the experiment, the mice fasted for 12 h were anesthetized and euthanized, and their serum was collected in endotoxin-free tubules. After labeling, the serum samples were centrifuged at 4 • C at 3000× g for 10 min and stored at −80 • C for further analysis. After serum collection, the liver and gut tissues of mice were removed and placed in pre-cooled normal saline for rinsing to remove the serum stuck to it separately, and the surface moisture was sucked out with filter paper. It was weighed and cut into pieces, one part was soaked in a preconfigured solution of 4% (v/v) DPEC (prepared in PBS), the other part was added 9 times 0.9% pre-cooled normal saline to prepare a 10% (w/v) homogenate, placed in a glass homogenizer for rapid grinding with an ice-cold bath, centrifuged at 12,000× g at 4 • C for 15 min, then the supernatant was taken and stored at −80 • C for later use.

Histopathological Analysis of Liver Tissues
Liver tissue fixed in 4% DPEC was further dehydrated, embedded in paraffin, sliced in 4 µm thickness, and stained with hematoxylin and eosin (H&E). Subsequent examination of the prepared slides was under a panoramic microscope (3D HITECH Panimal 250, Hungary) [35].

Determination of LPS in Gut Tissues
Gut LPS level was determined using ELISA assay kits obtained from Jiangsu Meimian industrial Co., Ltd. (Yancheng, China). The experimental procedures were quantified according to the supplier's instructions.

Analysis of Gut Microbiome
16S rRNA analysis was performed on frozen feces to analyze the gut microbial community structure and composition. Fecal total DNA was extracted and sequenced.
The universal primer pairs 338F and 806R were used to amplify the V3-V4 hypervariable region of the 16S rRNA gene [19]. Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Operational taxonomic units (OTUs) with 97% similarity cutoff were clustered using UPARSE version 7.1 [36], and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 [37] against the 16S rRNA database using a confidence threshold of 0.7. Assessment of alpha diversity by Chao richness, ACE index, Shannon and Simpson diversity indices using Sob's estimator [38]. Beta diversity at the OTU level was analyzed by principal component analysis (PCA) and non-metric multidimensional scaling anal-ysis (NMDS). The linear discriminant analysis effect size (LEfSe) method was used to identify differentially rich taxa in each treatment group, with the LDA score set at 4.0 [39].

Correlation Analysis
In order to further explore the relationship among His, PEA, β-be, and Iso, gut microbiota, microbial metabolites, and biomarkers mentioned above, Spearman correlation analysis was used to study the relationship between different gut microbiota and LPS, and LPS and biomarkers. If the absolute correlation coefficient is greater than 0.6 and p < 0.05, the correlation was considered significant. Heat maps were obtained using R software to visualize the results.

Statistical Analysis
The results are expressed as means ± SD (standard errors of the means) and analyzed using GraphPad Prism 8.0 program (GraphPad Software, San Diego, CA, USA). The data were evaluated using a one-way ANOVA analysis of variance by Ducan's test, when p < 0.05 was considered significant.