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3.6 Effects on intestinal histopathology in mice with alcoholic liver injury

As shown in the HE pathology in Figures 3 and 4, the intestinal villi in the small intestine of the mice in the normal group and normal administration group have a complete structure, are neatly arranged, complete and continuous, and the lamina propria gland cell structure is clear; compared with the normal group, the model The mice in the group were arranged disorderly; as shown in Figure 3, the microvilli of the small intestinal epithelial cells of the mice in the normal group and the normal administration group were longer, had normal structure, and were arranged neatly, completely and continuously; compared with the normal group, the microvilli in the model group were smaller The mice were disorganized; the microvilli were significantly shorter and disorganized; compared with the model group, the microvilli morphology of the mice in the CPhGs high-dose group was improved.

As shown in the AB-PAS pathology in Figures 3 and 4, compared with the normal group, the number of unemptied goblet cells in the jejunum of the model group was significantly reduced, and the secretion of acidic mucus protein was significantly increased; the jejunum and colon of the normal group were The number of unemptied goblet cells was the highest; the secretion of acidic mucus proteins in the jejunum and colon was reduced in the CPhGs high-dose group compared with the model group.

As shown in the immunohistochemical pathology of Muc2 in Figures 3 and 4, compared with the normal group, the expression of Muc2 in the jejunum and colon of mice in the model group was significantly increased; compared with the jejunum, the secretion of Muc2 in the colon changed more significantly. , while the jejunum is mainly concentrated in the deep crypts; compared with the model group, the expression of Muc2 in the jejunum and colon of mice in the CPhGs high-dose group was reduced.

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3.7 Effect of CPhGs on the Diversity and species distribution of intestinal flora in MMice with alcoholic liver injury

The number of ASVs was 300 in the normal group, 1395 in the normal dosing group, 445 in the model group, and 567 in the CPhGs high-dose group, see Enhanced Publication Additional Material. As shown in Table 4, compared with the normal group, the Shannon index of the model group was significantly reduced (P<0.05), and the Chao1 index

Compared with the model group, the Shannon index of the CPhGs high-dose group was significantly increased (P<0.01).

PCoA analyzes the Beta diversity index. Projecting the sample to the abscissa has an explanation of 35.70% of the bacterial flora between the groups, and projecting the sample to the ordinate has an explanation of 13.20% of the bacterial flora between the groups. The model group was far away from the normal group and normal administration group, and there were significant differences between the bacterial groups (P<0.01);

Compared with the model group, the bacterial flora in the CPhGs high-dose group showed significant changes (P < 0.01), see the supplementary material of the enhanced publication. As shown in Figure 5, at the genus level, the distribution of bacterial flora in the model group changed significantly; the composition of the bacterial flora in the normal group, normal dosage group, and CPhGs high-dose group was similar.

Table 4 Effect of CPhGs on Alpha diversity index of gut microbiota in ALD mice (𝑥̅± 𝑠, n=6)

Fig. 5 Effect of CPhGs on species distribution of gut microbiota in ALD mice at genus (x̅ ± s, n=6)

3.9 Correlation analysis of different intestinal flora of mice in each group

The total abundance of Akkermansia in Verrucomicrobia and Allobaculum in Firmicutes was the highest among all differential species; the abundance of Allobaculum was negatively correlated with the abundance of Akkermansia (r = -0.701, P<0.01);

Allobaculum genus abundance was associated with liver LBP levels (P＜0.01), serum IL-1β levels (P＜0.01), serum D-LA levels (P＜0.01), and serum AST, ALT, LPS, LBP, and TNF-α levels. (P<0.05) was positively correlated; the abundance of Akkermansia was related to

Serum LBP levels (P < 0.01), liver LBP and TG levels (P < 0.01), and serum ALT, AST, TG, IL-1β and LPS levels (P < 0.01) were negatively correlated, see Enhanced Publication Additional Material.

4. Discussion

This study found that CPhGs could improve liver function and hepatic steatosis and reduce serum inflammation and endotoxin levels in ALD mice. ALD has a wide range of manifestations. Among long-term alcohol drinkers, more than 80% of alcoholics will develop hepatic steatosis [6]. Currently, there is no prevention and treatment for ALD

with specific drugs. Recent studies have found that traditional Chinese medicine can delay the development of liver disease by regulating the balance of GM [18]. CPhGs are the indicator components of Cistanche deserticola, mainly including echinaceaside and verbascoside [19]. Modern pharmacological research has found that CPhGs have biological activities such as antioxidant, anti-inflammatory, and liver protection [15,20]. For example, verbascoside can inhibit immune liver injury induced by BCG combined with lipopolysaccharide [21]. The data of this study suggest that CPhGs can improve liver function and hepatic steatosis, and significantly reduce liver and serum TG levels. Moreover, CPhGs can also improve the intestinal barrier and intestinal flora homeostasis [22], mainly by promoting the growth of beneficial bacteria and regulating the level of mucus secretion by intestinal goblet cells.

Part of the effect of CPhGs in improving ALD is related to the regulation of intestinal mucus homeostasis. The intestine is the primary site of alcohol damage, and the intestinal barrier influences the role of GM in liver disease. Intestinal barriers include biological barriers (symbiotic flora), chemical barriers (intestinal mucus), physical barriers (top

epidermal tight junctions), and immune barriers (intestinal immune system) [23]. When exogenous substances break through the intestinal barrier, they flood into the liver through the hepatic portal vein, causing inflammatory reactions and liver damage [24]. There may even be a shift of bacterial flora, affecting liver metabolism and repair functions [25]. Intestinal mucosal morphological examination is the most direct evidence of intestinal barrier damage. This study used transmission electron microscopy to observe that the length of microvilli in the jejunal epithelial cells of mice in the model group was shortened and their arrangement was disordered. After CPhG intervention, the number of microvilli increased significantly, and the structure was relatively complete and neatly arranged. In addition, serum LPS and LBP can reflect serum intestinal endotoxin levels. Serum DAO and D-LA levels can reflect the integrity and damage of the intestinal mechanical barrier. LBP, as a parameter of total bacterial load, is the most accurate biomarker. DAO is a highly active intracellular enzyme in the villi of the intestinal mucosa, and D-LA is an anaerobic glycolytic product of intestinal commensal bacteria. Normally, serum levels of DAO and D-LA are low. When intestinal epithelial cells are damaged, DAO in the intestinal mucosa and D-LA in the intestinal lumen enter the bloodstream through the damaged site. Therefore, DAO and D-lactate water in serum

Ping can be used as an evaluation index for intestinal barrier damage [26].

The data of this study suggest that the levels of LPS, LBP, DAO, and D-LA in the model group increased significantly.

The above indicators were significantly reduced in the high, low, medium, and high dose groups of CPhGs. Therefore, CPhGs ameliorated intestinal barrier damage in ALD mice.

The effect of CPhGs on improving intestinal barrier damage in ALD mice is likely due to the regulation of intestinal mucus secretion. The stability of the intestinal mucus layer is critical to maintaining the integrity of the intestinal epithelium. The intestinal mucus protein is primarily Muc2, which is secreted by intestinal goblet cells. healthy gut

The goblet cells are not completely emptied to maintain continued mucus secretion. Studies have shown that alcohol intake can lead to mucosal damage and increased intestinal permeability [27,28]. Alcohol may dissolve lipids in the mucosa and reduce the hydrophobicity of the mucosal surface, leading to the loss of its barrier function [29]. In mice fed ethanol for 8 weeks, Muc2 expression was reduced in the ileum [30]. However, the effect of alcohol on the intestinal mucus barrier has not yet been studied. This study found that the acidic mucus secretion in the jejunum and colon of the model group was significantly increased, and the expression of Muc2 was also significantly increased. The microbial content in the intestine is much higher than that in other parts of the body. The excessive secretion of mucin by intestinal goblet cells is probably to enhance intestinal mucus flushing and inhibit the proliferation of potentially pathogenic bacteria in the intestine. Therefore, the number of goblet cells that store mucin in the intestine is depleted, and mucus increases in the intestinal lumen. In addition, relevant research has Now, that Muc2 deficiency ameliorates ALD in mice, and its protective effect depends on increased expression of antibacterial molecules [31,32]. Muc2 deficiency protects mice from high-fat diet-induced fatty liver disease [33]. This study found that CPhGs reduced intestinal mucus in ALD mice compared with the model group secretion volume. This regulatory effect may also help the antibacterial substances secreted by intestinal immune cells to exert antibacterial effects.

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This study mainly focused on the effects of CPhGs on intestinal barrier function and GM in ALD mice, so GM sequencing focused on the CPhGs normal administration group, model group, and CPhGs high-dose group. Due to the interaction between GM and the mucus layer, the presence of GM in the mucus layer can inhibit pathogenic bacteria.

Over-proliferation of bactericidal substances, some bacteria can also use the mucus layer to maintain the population. Among them, Akkermansia muciniphila muciniphila) can degrade intestinal mucin, and its mucin consumption and mucin regeneration by goblet cells can achieve a dynamic balance, thereby maintaining the stability of the mucus layer [34]. This study found that the abundance of the genus Akkermansia significantly decreased and mucus secretion increased significantly in the model group. After CPhG intervention, the abundance of the genus Akkermansia increased and the amount of mucus secretion decreased. The reasonable structure and abundance of the intestinal microbiota are crucial to suppressing the abundance of opportunistic pathogenic bacteria and maintaining the stability of the intestinal barrier [13]. Studies have found that transplanting fecal bacterial fluid from patients with severe alcoholic hepatitis into mice aggravated liver inflammation, increased intestinal permeability, and more frequent bacterial translocation [29]. It has been shown that co-colonization of the genus Allobaculum with Akkermansia muciniphila improves intestinal epithelial cell activation and colitis induced by the pathogenic bacteria Allobaculum genus [35]. And in people

The allobaculum genus was inversely associated with Akkermansia muciniphila in body-microbiota-associated mouse and human cohorts [34]. This study found that mice in the model group experienced excessive proliferation of the genus Allobaculum and a loss of abundance of the beneficial bacteria Akkermansia. Moreover, the Allobaculum genus was positively correlated with serum inflammation, endotoxin levels, etc., and the Akkermansia genus abundance was negatively correlated with the Allobaculum genus abundance. This study only evaluated intestinal mucus secretion from pathological sections, which has certain limitations. Moreover, there is a lack of experimental verification for the study of species relationships. However, what is clear is that CPhGs not only promote the proliferation of beneficial intestinal bacteria but also reduce the depletion of the number of goblet cells that store mucin, improving intestinal mucus barrier homeostasis. In summary, CPhGs can exert a partial protective effect on ALD by increasing the abundance of beneficial bacteria, reducing the abundance of potentially harmful bacteria, and reducing serum inflammation and endotoxin levels.

Conflict of Interest Statement: This article does not present any conflict of interest.

Reference

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