Increased plasma genistein after bariatric surgery could promote remission of NAFLD in patients with obesity

Abstract

Background: Bariatric surgery is associated with a positive effect on the progress of non-alcoholic associated fatty liver disease (NAFLD). Although weight loss is the obvious mechanism, there are also weight-independent mechanisms.

Methods: We collected blood samples from 5 patients with obesity before and 3 months after surgery and performed an LC-MS-based untargeted metabolomics test to detect potential systemic changes. We also constructed sleeve gastrectomy (SG) mice models. The plasma, liver and intestine samples were collected and analyzed by qPCR, ELISA and HPLC. Cohousing experiments and feces transplantation experiments were performed on mice to study the effect of gut microbiota. Genistein administration experiments were used to study the in vivo function of the metabolites.

Results: Plasma genistein (GE) was identified to be elevated after surgery. Both clinical data and rodent models suggested that plasma GE is negatively related to the degree of NAFLD. We fed diet-induced obese (DIO) mice with GE, and we found that there was significant remission of NAFLD. Both in vivo and in vitro experiments showed that GE could restrict the inflammation state in the liver and thus relieve NAFLD. Finally, we used co-housing experiments to alter the gut microbiota in mice, and it was identified that sleeve gastrectomy (SG) mice had a special gut microbiota phenotype, which could result in higher plasma GE levels. By feces transplantation experiment (FMT), we found that only feces from the SG mice (and not from other lean mice) could induce higher plasma GE levels.

Conclusion: Our studies showed that SG but not calorie restriction could induce higher plasma GE levels by altering the gut microbiota. This change could promote NAFLD remission. Our study provides new insights into the systemic effects of bariatric surgery. Bariatric surgery could affect remote organs via altered metabolites from the gut microbiota. Our study also identified that additional supplement of GE after surgery could be a therapy for NAFLD.

Keywords: bariatric surgery; genistein; gut microbiota; non-alcoholic fatty liver disease; obese; sleeve gastrectomy.

Figure 1

NAFLD is relieved after SG in patients with obesity. (A–C) BMI values, plasma AST, and TG before and 3 months after SG on patients with obesity. n = 5. (D) CT scan images of the liver in patients with obesity before and 3 months after SG surgery (three out of the five patients). (E) CT values of the liver before and 3 months after SG surgery on patients with obesity. n = 5. Wilcoxon test was used to compare the data before and after surgery. Data are presented as mean ± SEM, **p < 0.01, ***p < 0.001.

Figure 2

Plasma and local levels of genistein are increased after SG in patients with obesity. (A) Metabolite volcano map before and after SG in patients with obesity. (B) KEGG shows the affection of SG on metabolism in patients with obesity. (C) Analysis of the effect of SG on metabolism in patients with obesity. (D) Heatmap of relevant differential metabolites before and after SG. (E) Correlation analysis of the NAFLD degree and genistein abundance in patients with obesity. Spearman correlation was performed to study the relationship. Data are presented as mean ± SEM, **p < 0.01, ***p< 0.001.

Figure 3

Abundance of genistein is decreased in mice with obesity and could be restored by SG. (A–C) The concentration of genistein in the cecum, serum, and liver in different groups. n = 5 in each group. (D) The changes of body weight in each group. (E) The changes of plasma levels of GE in each group. The details of the group were introduced in the “Methods” part. ANOVA test was used to compare the differences among the groups. Data are presented as mean ± SEM, *p < 0.05.

Figure 4

Genistein could relieve NAFLD and decrease the inflammation status in the liver. (A, B) The infiltration of macrophages (marked by F4/80) and lipid accumulation were detected by immunohistochemistry in the liver tissue of mice from different age-matched groups. ND-control: normal mice fed with a normal diet. DIO: diet-induced obese mice. DIO+GE: DIO mice fed with HFD containing 2% GE for 8 weeks. Scale bar = 50 μm. (C). HE staining of livers from SG and SG+ GE mice. Scale bar = 50 μm (D, E). Serum AST and TG concentrations in SG and SG + GE mice. The DIO mice received and were fed with HFD (the SG group) or HFD+0.2% GE (the SG+GE group) for 8 weeks. n = 5 in each group. Mann–Whitney U test was used to analyze the differences between the two groups. Data are presented as mean ± SEM, *p < 0.05. The P value in the image used in Figures 4E is equal to 0.0833, less than 0.05.

Figure 5

Genistein could reduce pro-inflammatory cytokine production and chemotactic migration in macrophages in the liver. (A–C) The mRNA levels of TNF-α, IL-1β, and MCP-1 in the liver from different groups, tested by qPCR. (D–F) The protein levels of levels of TNF-α, IL-1β, and MCP-1 in the liver from different groups, tested by ELISA. The age-matched female mice received 6 weeks of normal diet (Control), HFD diet (HFD), and GE diet (HFD+0.2%GE). n = 5 in each group. (G–I) RAW 264.7 cells were stimulated with palmitic acid (Pal) followed by LPS, with or without addition of varying doses of genistein. The negative control (NC) group was only treated with the vehicle, dimethylformamide (DMF). Changes in the expression of TNF-a, IL-1β, and MCP-1 were determined using qPCR. (J) Genistein inhibits BMDM migration toward MCP-1. BMDMs were incubated with MCP-1 in a Transwell with vehicle (DMF) or different doses of genistein. The chemotactic index (CI) for a treatment condition was calculated as the ratio of average number of migrated cells in the treatment group relative to the control group (incubated in medium only). ANOVA test was used to compare the differences among the groups. Data are presented as mean ± SEM, * stands for p < 0.05, ** stands for p < 0.01 and *** stands for p < 0.001, compared with the negative control (NC) group. For G-I, # stands for the GE stimulation group compared with the Pal+LPS stimulation group, #p < 0.05, ##p < 0.01, ###p < 0.001.

Figure 6

Transplantation of feces from SG mice could induce GE abundance in recipient mice. (A–C) Genistein concentrations in cecum, serum, and liver in different groups. n = 6. DIO-SG group: DIO mice underwent SG and fed alone in one cage. DIO-SG+DIO cohousing group: DIO mice underwent SG and cohoused with DIO mice (3 + 3 in one cage) for 6 weeks. (D–F) Genistein concentrations in cecum, serum, and liver in different groups. n = 5. There are four groups, in each group the normal-diet-fed mice are recipient mice and received oral gavage of feces from SG mice (DIO-SG fecal group), DIO mice (DIO fecal group), and pair-weight mice (DIO-pair-weight fecal group). The control group contains five mice which were fed with a normal diet and did not receive oral gavage. ANOVA test was used to compare the differences among the groups. Mann–Whitney U test was used to analyze the differences between the two groups. Data are presented as mean ± SEM, ** stands for p < 0.01, and *** stands for p < 0.001.