Protective effect of Zhizi Huangqi Shanzha formula (栀子黄芪山楂方) on aflatoxin poisoning in mice and its effect on intestinal flora

Abstract

OBJECTIVE

To evaluate the protective effect of Zhizi Huangqi Shanzha formula (栀子黄芪山楂方, ZHSF) on aflatoxin-induced liver injury.

METHODS

The protective effect of ZHSF on the aflatoxin-induced liver injury was evaluated by histological observation, blood cell analysis, evaluation of liver function and immunity, and gut microbiota analysis.

RESULTS

ZHSF can significantly up-regulate the percentage of lymphocytes and eosinophils in the blood of Aflatoxin B1-intoxicated mice, down-regulate the levels of serum aspartate aminotransferase, alanine aminotransferase, and malondialdehyde, and recover the liver tissue structure. Aflatoxin poisoning induces a variation of the intestinal flora of mice, and ZHSF may recover the variation of intestinal flora induced by Aflatoxin B1. Cluster analysis showed that the intestinal flora of mice in the intervention group was more similar to that of the control group. Correlation analysis showed that Lachnospiraceae, Desulfovibrio, and Lactobacillus may be the key flora for the pharmacological effects of ZHSF.

CONCLUSIONS

ZHSF may protect against aflatoxin-induced liver damage, improve immunity, and inhibit oxidative stress by regulating the composition and relative abundance of intestinal flora, which makes it a promising liver-protective candidate drug.

1. INTRODUCTION

Aflatoxins are a class of mycotoxins produced by some strains of the moulds Aspergillus parasiticus and Aspergillus flavus. Aflatoxins are a group of structurally similar furanocoumarins, mainly produced by some strains including Aspergillus parasiticus and Aspergillus flavus. Chemically, the structures of aflatoxins consisted of an oxanonaphthalenone and a bifuran ring, the oxanonaphthalenone being associated with carcino-genesis, and the bifuran ring being the basic toxin structure, and the oxanonaphthalenone enhancing the toxicity of the bifuran ring. Currently, the most common aflatoxins are Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1), Aflatoxin G2 (AFG2), Aflatoxin M1 (AFM1), and Aflatoxin M2 (AFM2).¹ AFB1, AFB2, AFG1, and AFG2 are usually naturally occurring, while AFM1 and AFM2 are formed by the metabolic hydroxylation of AFB1 and AFB2 in vivo. Besides, The AFB1 is the predominant form and the most toxic, being far more hazardous than cyanide, arsenic, and organic pesticides,^2,⇓-4 and it was classified as a human carcinogen by the Agency for Research on Cancer as early as 1993.

AFB1 exists in moldy soil, plants, and various nuts, especially peanuts and walnuts. AFB1 is also often found in moldy beans, dairy products, and cereals, which could easily contaminate poorly stored food or feeds such as peanuts, corn, rice, wheat, and soybean.⁵ The toxicity of AFB1 on the body is mainly presented in three ways. First, the main target of AFB1 toxicity is the liver,⁶ where it is metabolized by cytochrome P450 (CYP450) enzymes to produce poisonous metabolites AFB1-exo-8,9-epoxide, contributing to the development of liver disease.⁷ And AFB1 also causes hepatocyte necrosis, bile duct hyperplasia, liver fibrosis, and even hepatocellular carcinoma, and is recognized as one of two important factors closely related to the development of primary hepatocellular carcinoma. Second, AFB1 has immunosuppressive toxicity and inhibits the body's ability against secondary infections caused by bacteria, fungi, and parasites.⁸ AFB1 also inhibits the function and reduces the number of lymphocytes, and inhibits phagocytosis, complement and natural killer cell activity. Third, AFB1 can damage multiple organs including liver,⁹ kidney,¹⁰ heart,¹¹ and spleen¹² through oxidative stress. It has been reported that after restoring the balance of oxidative stress and antioxidant systems, the pathological variation of lungs, spleen, and liver tissues and cell apoptosis of human venous endothelial cells or bovine mammary epithelial cells are reduced^13,⇓-15 through decreasing the levels of reactive oxygen species, hydrogen peroxide and malondialdehyde (MDA), and increasing the total antioxidant capacity and antioxidant enzyme activity.¹⁶

According to the theory of Traditional Chinese Medicine and modern pharmacological research. The effectiveness of Zhizi (Fructus Gardeniae) includes dispelling fire and removing irritation, clearing heat and dampness, cooling the blood and detoxifying. And modern pharmacological research shows that it has many effects including hepatoprotective, cholagogic, hypotension, sedation, hemostasis, and detumescence.¹⁷ It is commonly used in the treatment of icterohepatitis, hypertension, and diabetes. However, Zhizi (Fructus Gardeniae) was not suitable for chronic diarrhea with spleen deficiency syndrome because of its bitter-cold and injurious to the stomach. Therefore, it should be used in combination with tonics. The effectiveness of Huangqi (Radix Astragali Mongolici) includes invigorating Qi for ascending, consolidating superficies for arresting sweating, promoting the production of fluid for replenishing blood, and modern pharmacological research indicated that it can enhance the immune function of the body.¹⁸ Thus, Huangqi (Radix Astragali Mongolici) was chosen to relieve the bitter-cold nature of Zhizi (Fructus Gardeniae) from damaging the organism. Shanzha (Fructus Crataegus Pinnatifidae) plays a role in strengthening the spleen and appetite, eliminating food stagnation, while modern pharmacological research shows that the total polyphenols and total flavonoids it contains can scavenge 1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) free radicals and have strong antioxidant effects.¹⁹ In theory, the combination of hepatoprotective and cholagogic of Zhizi (Fructus Gardeniae), invigorating Qi and immunity-enhancing effects of Huangqi (Radix Astragali Mongolici), and spleen-enhancing effects of Shanzha (Fructus Crataegus Pinnatifidae) could effectively alleviate the liver damage, immune suppression and oxidative stress caused by AFB1. What’s more, Zhizi (Fructus Gardeniae), Shanzha (Fructus Crataegus Pinnatifidae), and Huangqi (Radix Astragali Mongolici) were contained in many Chinese patent medicines including Yinqi Ganfu granule (茵芪肝复颗粒), Yuye Jin Wan (玉液金丸), and Fufang Xuanju Yiqi Pian (方玄驹益气片), of which corroborate the compatibility of Zhizi (Fructus Gardeniae), Shanzha (Fructus Crataegus Pinnatifidae), and Huangqi (Radix Astragali Mongolici).

In this study, Zhizi Huangqi Shanzha formula (栀子黄芪山楂方), composed of Zhizi (Fructus Gardeniae), Shanzha (Fructus Crataegus Pinnatifidae), and Huangqi (Radix Astragali Mongolici), were used to investigate their protective effects on aflatoxin-induced liver injury in mice based on the above theoretical basis. and to analyze the variation in their intestinal flora and metabolites to explore their possible mechanisms of action.

2. MATERIALS AND METHODS

2.1. Chinese crude drug and reagents for testing

Zhizi (Fructus Gardeniae), Shanzha (Fructus Crataegus Pinnatifidae), and Huangqi (Radix Astragali Mongolici) were provided by Yinghuitang Pharmaceutical Co. Ltd., Bozhou, Anhui, China in October 2021. Ethanol (95%), chromatographic grade acetonitrile, formic acid, and methanol purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Double-distilled water was freshly prepared using an automatic double-water distillation machine. Aflatoxin B1 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Silibin Meglumine Tablets (水飞蓟宾葡甲胺片) (National Medicine Permit No. H32026145) were purchased from Jiangsu Zhongxing Pharmaceutical Co., Ltd. (Zhenjiang, China). Alanine aminotransferase (ALT) (C009-1-1), aspartate aminotransferase (AST) (C010-1-1), malondialdehyde (MDA), and superoxide dismutase (SOD) detecting kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Mouse immunoglobulin G (IgG) and immunoglobulin G (IgM) enzyme linked immunosorbent assay (ELISA) kits were from Multisciences (Lianke) Biotech., Co., Ltd. (Hangzhou, China). Blood test reagents were purchased from Shenzhen Dymatic Biotechnology Co., Ltd. (Shenzhen, China). Hematoxylin-eosin stain purchased from Solaribio Biotechnology Co., Ltd. (Shanghai, China).

2.2. Preparation of ZHSF

A 100 g Zhizi (Fructus Gardeniae) was extracted with eight-fold 75% ethanol twice under reflux for 2 h. Then, A 50 g Huangqi (Radix Astragali Mongolici) was extracted with eight-fold 75% ethanol twice under reflux for 2 h. and another 50 g Huangqi (Radix Astragali Mongolici) was extracted with water twice under reflux for 2 h. Besides, 100 g Shanzha (Fructus Crataegus Pinnatifidae) was also extracted with eight-fold 75% ethanol twice under reflux for 2 h. After that, the extract was filtered through medium-speed filter paper with a pore size ranged ranging from 50 to 80 μm. After filtration, the combined solution was concentrated under reduced pressure at 45 ℃ to remove the ethanol. And they were mixed with Cyclodextrin, and disintegrating starch to obtain the compound Chinese Traditional Medicine ZHSF, of which raw herb content was 1 g/g.

2.3. Experimental animals breeding and experiment design

Female mice were chosen for this study because male mice are aggressive and may interfere with the results of the experiment. Concretely, 5-week female Institute of Cancer Research mice were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China. Mice were adaptively bred for one week. Sterile water and irradiated feed were given to mice. The mice used in this study weighed between 16 and 18 g. Mice were kept under temperature [(24 ± 1) ℃], humidity (50% ± 10%), and a 12/12 h light/dark cycle. This study was conducted based on the guidelines established by the Institute for Experimental Animals of West Anhui University and the protocol was approved by the University Local Committee on the Ethical Use of Animal Experiments [2022-E(r)-053]. A total of 30 mice were randomly divided into six groups including the Control group, Model group, Positive group, and ZHSF treatment group (1.5, 3, 6 g/kg). The dosage of ZHSF was determined according to the composition and amount of raw herbal drug and the dosage of raw herbal drug.

Normal control group: mice were orally administered (p.o.) with saline for 10 d. Model group: Mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with saline for 7 d before the sample harvesting. Positive control group: Mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with Silibin Meglumine for 7 d before the sample harvesting. AFB1 + ZHSF (1.5, 3.0, or 6.0 g/kg) group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with ZHSF (1.5, 3.0, or 6.0 g/kg) for 7 d before the sample harvesting.

2.4. Sample collected

Mice were anesthetized with 0.1 mL 0.3% pentobarbital sodium solution, and then the anticoagulated and non-anticoagulated blood were collected. Then, the mice were euthanatized using the carbon dioxide inhalation asphyxiation method. The non-anticoagulated blood was placed at 37 ℃ for 2 h and centrifugated at 3500 rpm for 10 min to obtain the serum. And the liver, cecum contents were obtained under sterile conditions. The Liver tissue was used for H.E staining to observe liver tissue structure. Cecum contents were used for 16s rDNA sequencing analysis

2.5. Histological observation

Liver tissue was fixed with 4% paraformaldehyde, and the fixed tissue was subsequently rinsed with running water for 12 h, followed by dehydration two times with gradient ethanol (70%-80%-95%-100%). Then, the tissue was sequence transparent in ethanol-xylene, xylene Ⅰ, and xylene Ⅱ. After that, the tissue was embedded in paraffin and cut into pieces with 5 μm thicknesses. The tissue pieces were spread evenly on a slide and baked at 66℃for 30 min. Then, the microsections, stained with hematoxylin and eosin (HE), were observed using an MSHOT60 microscope (Guangzhou, China).

2.6. Analysis of blood cell

The neutrophils, lymphocytes, granulocytes, eosinophils, and basophils in whole blood were measured using Dymind Animal Blood Cell Analyzer (Shenzhen, China). The values were expressed as the percentage of blood cells.

2.7. Serum ALT and AST

The serum levels of ALT and AST in each group of mice were detected using the ALT and AST detecting kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The modified procedure based on the reagent kit instructions was listed as follows. A 5 μL sample and 25 μL substrate solution were added in assay wells distributed in a 96-well plate, a sealing touch, and shaken lightly. And after that, the plate was incubated for 30 min at 37 ℃. Afterward, a 5 μL sample and 25 μL 2,4-dinitrophenylhydrazine solution were added to control wells distributed in a 96-well plate, apply a sealing touch and shake lightly. After that, the plate was incubated for 20 min at 37 ℃. After that, 240 μL of 0.4 M NaOH solution was added to each well. The optical density value of each well was measured at 550 nm and the ALT and AST levels of each sample were calculated according to the instructions.

2.8. Serum IgG and IgM antibody

The serum IgG and IgM antibodies were measured using a commercial ELISA kit as previous research described.²⁰

2.9. Serum MDA and SOD

The serum levels of MDA and SOD in each group of mice were detected using the MDA and SOD detecting kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). And the specific steps were conducted according to instructions of detecting kits.

2.10. Gut microbiota analysis

Feces were collected after the intervention. Then, the DNA was extracted using QIAam DNA Stool Mini Kit, produced by QIAGEN (Nordrhein-Westfalen, German). Then, it was amplified at the V4 region of the 16s ribosomal RNA (rRNA) subunit gene, and 250 nucleotides (nt) Illumina sequencing reads were generated. 16s rRNA gene amplicon sequencing and data analysis were performed as previous research described.²¹ Alpha diversity of gut microbiota was expressed by Ace, Chao1 and Shannon index, and Beta diversity was expressed by principal co-ordinates analysis. The linear discriminant analysis effect size (LEFSe) analysis was conducted to screen the bacteria with significant differences at each taxonomic level of three groups. And the possible functional flora was screened using correlation analysis.

2.11. Data statistics

The quantitative data were expressed as mean ± standard deviation ($\overline{x}\pm s$). The difference analysis compared to the model group was conducted using SPSS Statistics 22.0 (IBM Corp., Armonk, NY, USA) in accordance with Student's t-tests. P-values less than 0.05 were statistically significant differences.

3. RESULTS

3.1. Effect of ZHSF on the blood cell composition of mice poisoned with AFB1

As shown in Figure 1, it can be found that the percentage of neutrophils and monocytes in the model group was significantly increased, which indicated that AFB1 may cause an inflammatory response. After the intervention of ZHSF, the percentage of blood neutrophils and monocytes in the low and medium dose groups was significantly decreased compared with the model group, which means that ZHSF alleviating the inflammatory response caused by AFB1. The percentage of lymphocytes and eosinophils in the model group were decreased significantly compared with that in the control group (P<0.05), while the percentage of lymphocytes and eosinophils in the ZHSF group with a low dose increased significantly compared with that in the model group (P<0.05, P<0.01). The above results suggest that AFB1 can cause a decrease of immunity in mice, and the ZHSF can effectively enhance the immunity of aflatoxin poisoned mice.

Figure 1. Effect of ZHSF on the blood cell composition of mice poisoned with AFB1.

A: percentage of lymphocyte in the groups. B: percentage of eosinophils in the groups. C: percentage of monocyte in the groups. D: percentage of neutrophils in the groups. Control group: mice were orally administered (p.o.) with saline for 10 d. Model group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with saline for 7 d before the sample harvesting. AFB1 + ZHSF (1.5 g/kg) group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with ZHSF (1.5 g/kg) for 7 d before the sample harvesting. AFB1 + ZHSF (3.0 g/kg) group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with ZHSF (3.0 g/kg) for 7 d before the sample harvesting. AFB1 + ZHSF (6.0 g/kg) group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with ZHSF (6.0 g/kg) for 7 d before the sample harvesting. Blood routine analysis was used to analyze the blood cell composition in control group, model group, positive group and ZHSF groups. ZHSF: Zhizi Huangqi Shanzha formula; AFB1: aflatoxin B1; Lym: lymphocyte; Mon: monocyte; Eos: eosinophils; Neu: neutrophils. All data was measured by one-way analysis, and Newman-Keuls test was performed for inter-group comparisons. All data was presented as mean ± standard deviation (n = 5). ^aP<0.05 compared to the model group.

3.2. ZHSF effectively improve liver function of mice poisoned with AFB1

As shown in Figure 2, it can be seen that the liver tissue in the control group had intact hepatic lobule structure and radiating hepatic cords and hepatic blood sinusoidal structure around the central vein, while the hepatic lobule structure of the model group almost disappeared and the hepatic cords were disordered. These results again confirmed that AFB1 could cause obvious damage to the liver tissue. The structural features of hepatic lobules could be clearly observed in the low-dose group of ZHSF, while there was not an obvious improvement in the middle-dose and high-dose groups of ZHSF. The serum AST and ALT of the model group were significantly higher than those of the control group (P<0.01, P<0.001), suggesting that the liver injury model induced by AFB1 was successful. The serum AST and ALT of mice in the positive control group and the low dose of ZHSF were significantly lower than those in the model group (P < 0.05, P < 0.01) (Figure 2B). The serum MDA level of the model group was significantly higher than that of the control group (P < 0.001), while the serum MDA in the positive and ZHSF treatment group was significantly lower than that in the model group. The serum SOD level of the model group was significantly lower than those of the control group (P < 0.001), while the serum SOD in the positive and ZHSF treatment group was significantly higher than that in the model group (Figure 2C).

Figure 2. Effect of ZHSF on liver function and oxidative stress of AFB1 poisoning mice.

A: hematoxylin-eosin staining was used to analyze morphological and histological of liver in control group, model group, positive group and ZHSF groups. A1: Control group; A2: Model group; A3: Positive group; A4: ZHSF (1.5 g/kg) group; A5: ZHSF (3.0 g/kg) group; A6: ZHSF (6.0 g/kg) group. B: result of ALT; C: result of AST; D: result of SOD; E: result of MDA. Control group: Mice were orally administered (p.o.) with saline for 10 d. Model group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with saline for 7 d before the sample harvesting. AFB1 + ZHSF (1.5 g/kg) group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with ZHSF (1.5 g/kg) for 7 d before the sample harvesting. AFB1 + ZHSF (3.0 g/kg) group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with ZHSF (3.0 g/kg) for 7 d before the sample harvesting. AFB1 + ZHSF (6.0 g/kg) group: mice were orally administered (p.o.) with AFB1 of 0.4 mg/kg from the first day to the third day and orally administered (p.o.) with ZHSF (6.0 g/kg) for 7 d before the sample harvesting. ZHSF: Zhizi Huangqi Shanzha formula; AFB1: aflatoxin B1; ALT: alanine aminotransferase; AST: aspartate aminotransferase; SOD: superoxide dismutase; MDA: malondialdehyde. All data was measured by one-way analysis, and Newman-Keuls test was performed for inter-group comparisons. All data was presented as mean ± standard deviation (n = 5). ^aP<0.05 compared to the model group.

3.3. ZHSF increase IgG and IgM antibody titers in aflatoxin B1 poisoned mice

As presented in Table 1, the content of serum IgG and IgM in the model group was significantly lower than those in the control group (P < 0.01, P < 0.001), suggesting that AFB1 can cause immunosuppression. The serum IgG and IgM levels of mice in the positive control group and the low-dose ZHSF group were significantly higher than those in the model group (P < 0.05, P < 0.01). The above results suggest that ZHSF can effectively improve the immune deficiency caused by AFB1.

Table 1.

Effect of ZHSF on the content of serum IgG and IgM mice poisoned with AFB1

Item	Control group	Model group	Positive group	ZHSF (g/kg)
				1.5	3	6
IgG (μg/mL)	1556±65a	1259±118	1625±96a	1508±31a	1323±106a	1325±144
IgM (μg/mL)	223±37a	92±21	129±31	159±13a	117±16a	128±12a

Notes: the enzyme linked immunosorbent assay was used to measure the content of IgG and IgM in in control group, model group, positive group and ZHSF groups. IgG: Immunoglobulin G; IgM: Immunoglobulin M. All data was measured by one-way analysis, and Newman-Keuls test was performed for inter-group comparisons. All data was presented as mean ± standard deviation (n = 5). ^aP<0.05 compared to the model group.

3.4. ZHSF can effectively improve the intestinal flora structure of aflatoxin-poisoned mice

As shown in supplementary Figures 1-4, AFB1 reduces the abundance of HT002, Bacteroides, Lachnospiraceae, Clostridiales, Colidextribacter, Eubacterium, Lachnos piraceae_NK4A136_group, Peptococcaceae_un-classified, Firmicutes_unclassified, Ruminococcaceae_ unclassified, Anaerotignum, Streptococcus, Monoglobus, Clostridium, Eubacterium_xylanophilum Ligilactobacillus, Muribaculaceae, Clostridia_UCG−014, while the abundance of these florae was increased by ZHSF. Besides, the relative abundance of Desulfovibrio, RF39_unclassified, Alistipes, Erysipelotrichaceae_ unclassified, Ruminococcus, Alloprevotella, Mucispirillum, Erysipelatoclostridium, Enterorhabdus, Candidatus, Saccharimonas, Lactobacillus, Akkermansia were elevated by AFB1, and ZHSF decreases the abundance of above species except for Mucispirillum and Erysipelatoclostridium (supplementary Figures 1, 2). The above results indicate that ZHSF can effectively restore the disorder of intestinal flora in mice caused by AFB1. In addition, the results of cluster analysis also showed that the intestinal flora of mice in the intervention group was more similar to that of the control group.

To screen the core functional flora, the LEfSe analysis and correlation analysis were conducted (supplementary Figure 3). The results of LEfSe analysis showed that the dominant flora in the ZHSF intervention group included HT002, Eubacteriaceae, Eubacterium, Lachnospiraceae, Olsenella, Atopobiaceae, Coriobacteriales, Olsenella_s p_F0206, Ileibacterium, Ileibacterium_unclassified, Clostridium, Moraxellaceae, Acinetobacter, Mesorhizobium, Dubosiella, Mesorhizobium, Dubosiella, Acinetobacter, Phyllobacteriaceae. And the Methylobacterium,_ Methylorubrum_unclassified, Chloroplast_unclassified, Chloroplast, Cyanobacteria, and Chloroplast_ unclassified were dominant flora in the model group. The correlation analysis between the abundance of TOP30 groups and the level of AST, ALT, IgG, and IgM were conducted, respectively (supplementary Figure 4). The results showed a significant negative correlation between AST and Lachnospiraceae_NK4A136_group (P < 0.01). The relative abundance of Lactobacillus was a positive correlation with the level of serum AST (P < 0.01). Besides, the relative abundance of Desulfovibrio and Lactobacillus had a positive correlation with the level of serum ALT (P < 0.05). What’s more, Serum IgG and IgM levels were also significantly correlated with the relative abundance of Lachnospiraceae_NK4A136, Desulfovibrio, and Lactobacillus. The results of the above analysis suggest that Lachnospiraceae_ NK4A136_group, Desulfovibrio, and Lactobacillus may be the key flora for the pharmacological effects of ZHSF, which should be studied in depth in the next study.

4. DISCUSSION

Aflatoxin leads to a wide range of adverse symptoms, including immunosuppression, hepatic impairment, and oxidative stress. Zhizi (Fructus Gardeniae), Shanzha (Fructus Crataegus Pinnatifidae), and Huangqi (Radix Astragali Mongolici) were combined to investigate their protective effect on aflatoxin poisoning based on traditional Chinese medicine and modern pharmacological research. ZHSF can significantly up-regulate the percentage of lymphocytes and eosinophils in the blood of AFB1-intoxicated mice, down-regulate the levels of serum AST, ALT, SOD, and MDA in AFB1-intoxicated mice, and recover the liver tissue structure according to the results of blood cell analysis, hematoxylin-eosin staining of liver tissue, biochemical parameters and serum immunoglobulin testing. ZHSF can effectively improve immunosuppression, oxidative stress, and liver damage caused by AFB1.

In recent years, the concept of the "gut-liver axis" has led to a new awareness of the relationship between intestinal flora and liver disease.²² Firstly, the liver and the intestine are closely related as they share the same embryonic origin and 70% of the blood in the liver comes from the intestine, of which contains metabolites from digestion and intestinal microbial.²³ The intact intestinal mucosa can prevent the migration of most intestinal bacteria and their products. Once the integrity of the intestinal tract was destroyed, the barrier function of the intestinal tract would be damaged by dysbiosis of intestinal flora and overgrowth of the intestinal bacteria. Then, the products of the intestinal bacteria may cause liver damage due to entering the liver through the hepatic portal vein.²² What’s more, dysbiosis of the intestinal flora is an important pathway in the development and progression of autoimmune liver disease,²⁴ alcoholic liver disease,²⁵ metabolic-related fatty liver disease, viral hepatitis,²⁶ cirrhosis, and hepatocellular carcinoma.²⁷

To investigate the action mechanism ZHSF on the protection of mice from aflatoxin poisoning. The composition of intestinal flora in the cecum contents of mice in the control, model, and ZHSF intervention groups were analyzed using 16S rDNA sequencing, respectively. The results showed that ZHSF changed the composition of the intestinal flora induced by aflatoxicosis. The abundance and species diversity of the flora of the mice treated with ZHSF were higher than those in the control and model groups. As a result, the Lachnospiraceae_NK4A136_group, Desulfovibrio, and Lactobacillus may be the key flora for the pharmacological effects of ZHSF. Genus Lachnos-piraceae was present in the gut of most healthy people, and it is a potentially beneficial bacterium, participating in the metabolism of a wide range of carbohydrates, especially for a complex dietary fiber and prebiotic in fruits and vegetables. Besides, it is highly capable of fermenting to produce acetic acid and butyric acid, which could provide energy to the host. This result was in general agreement with our previous finding that a positive correlation between the relative abundance of Lachnospiraceae and serum immunoglobulin levels. Surprisingly, a negative correlation between liver function indicators and the relative abundance of Lachnospiraceae, which may be due to a large number of volatile fatty acids produced by Lachnospiraceae which increases the metabolic load of the liver.²⁸ Besides, the increased abundance of the genus Desulfovibrio contributes to the improvement of liver function, but it is detrimental to the recovery of immune function. Desulfovibrio is known to produce high levels of hydrogen sulfide, which may increase the risk of recurrence of ulcerative colitis and Crohn's disease, but there was also some research indicating that it could promote gastrointestinal healing.²⁹ Therefore, there may be a bidirectional regulation of Desulfovibrio's function, and the exact mechanism needs to be further investigated.

Although the efficacy of ZHSF was confirmed in this study, the scientific basis of the formula, the ingredients that play a key role in the formula, the mechanism of ZHSF regulate the intestinal flora, and the validation of intestinal flora function was not carried out in-depth in this study because of the extremely complex active ingredients contained in ZHSF. It was reported in previous research that geniposide in Zhizi (Fructus Gardeniae), flavonoids, saponins and polysaccharides contained in Huangqi (Radix Astragali Mongolici), and flavonoids contained in Shanzha (Fructus Crataegus Pinnatifidae) can have a better protective effect against liver injury caused by various factors. And these components may be the main active ingredients of ZHSF. Therefore, it is proposed to confirm the mechanism of action and targets of ZHSF for the treatment of aflatoxin poisoning through proteomics, metabolomics, and composition analysis, combined with cellular model construction.

In conclusion, ZHSF may protect against aflatoxin-induced liver damage, improve immunity, and inhibit oxidative stress by regulating the composition and relative abundance of intestinal flora, of which makes it a promising liver-protective candidate drug.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.cn.

Funding Statement

Supported by the Provincial Natural Science Foundation of Anhui Province-Quantitative Constitutive Effect and Mechanism of Action of Dendrobium Polysaccharides against Tumours based on the Tumour Microenvironment (2208085QH273), and Natural Science Research Project for Anhui Universities-mechanism of Anti-bacterial Diarrhea Effect of Probiotic-fermented Portulaca Oleracea in Targeting and Regulating Amino acid Metabolism of Intestinal Flora and Screening of Anti-tumor Active Fraction of Paris polyphylla and its Effect on the Tumour Immune Microenvironment (2023AH052634, 2022AH051668)