Yinchen Linggui Zhugan decoction ameliorates high fat diet-induced nonalcoholic fatty liver disease by modulation of SIRT1/Nrf2 signaling pathway and gut microbiota - PubMed (original) (raw)

. 2022 Dec 12:13:1001778.

doi: 10.3389/fmicb.2022.1001778. eCollection 2022.

Tangyou Mao 2, Zhongmei Sun 1 2, Lei Shi 2, Xiao Han 1 2, Yang Zhang 1 2, Xiaosi Zhang 1 2, Jiali Wang 1 2, Juncong Hu 1 2, Liming Zhang 1 2, Junxiang Li 2, Haixiao Han 2

Affiliations

Yinchen Linggui Zhugan decoction ameliorates high fat diet-induced nonalcoholic fatty liver disease by modulation of SIRT1/Nrf2 signaling pathway and gut microbiota

Hui Jiang et al. Front Microbiol. 2022.

Abstract

Yinchen Linggui Zhugan decoction (YLZD) is an effective and classical traditional herbal prescription for treating the nonalcoholic fatty liver disease (NAFLD) and has been proven to be effective in the regulation of lipid metabolism disorder and attenuate inflammation for a NAFLD rat model. However, the exact underlying mechanism has not been elucidated. In the current study, a NAFLD rat model was established using a high-fat diet (HFD) for 10 weeks, followed by YLZD treatment with 1.92 g/kg/day for 4 weeks to explore the mechanisms of YLZD. Our results showed that YLZD decreased the hepatic lipid deposition, restored the liver tissue pathological lesions, inhibited the expression of oxidative stress, and decreased the inflammatory cytokines levels. Meanwhile, the genes and proteins expressions of SIRT1/Nrf2 signaling pathway together with downstream factors including HO-1 and NQO1 were elevated in the YLZD treated NAFLD rats. For further elaborating the upstream mechanism, short-chain fatty acids (SCFAs) in serum and feces were measured by liquid chromatograph mass spectrometer and gas chromatograph mass spectrometer, and the differences in gut microbiota of rats in each group were analyzed through high-throughput sequencing of 16S rRNA. The results demonstrated that the contents of butyric acid (BA) and total SCFAs in YLZD-treated NAFLD rats were significantly increased in serum and feces. 16S rRNA sequencing analysis illustrated that YLZD intervention led to a modification of the gut microbiota composition, with a decrease of Oribacterium, Lactobacillus and the ratio of Firmicutes/Bacteroides, as well as the increase in SCFAs-producing bacteria such as Christensenellaceae, Clostridia, Muribaculaceae, and Prevotellaceae. Spearman rank correlation analysis indicated that BA and total SCFAs were negatively co-related with oxidative stress-related factors and inflammatory cytokines, while they were positively co-related with SIRT1/Nrf2 pathway related genes and proteins. Furthermore, in vitro study confirmed that BA effectively reduced oxidative stress by activating SIRT1/Nrf2 signaling pathway in L02 cells. Together, the present data revealed YLZD could ameliorate HFD-induced NAFLD in rats by the modulation of SIRT1/Nrf2 signaling pathway and gut microbiota.

Keywords: Yinchen Linggui Zhugan decoction; gut microbiota; metabolites; nonalcoholic fatty liver disease; short-chain fatty acids; spearman rank correlation analysis.

Copyright © 2022 Jiang, Mao, Sun, Shi, Han, Zhang, Zhang, Wang, Hu, Zhang, Li and Han.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1

Figure 1

YLZD improved the general status and mitigated hepatic lipid deposition. (A) Experimental design, (B) daily food intake, (C) body weight change, (D) Liver index = Liver weight/Body weight × 100, (E) Epididymal fat index = Epididymal fat weight/Body weight × 100, (F–H) The level of liver FFA, TG and TC, respectively, and (I) Representative images of HE and oil red staining livers (magnification, ×200). Control group, normal rats treated with distilled water; HFD group, HFD-induced liver injury rats treated with distilled water; YLZD group, HFD induced liver-damaged rats treated with YLZD 1.92 g/kg/day for 4 weeks. Values are represented as mean ± SEM (n = 6 rats/group). ##p < 0.01, #p < 0.05 versus the Control group; **p < 0.01, *p < 0.05 versus the HFD group.

Figure 2

Figure 2

YLZD alleviated oxidative stress damage and reduced NAFLD-related systemic inflammation. Liver homogenate levels of (A) T-AOC, (B) SOD, (C) GSH-Px, (D) ROS and (E) MDA, respectively. Serum levels of (F) TNF-α, (G) IL-6 and (H) IL-1β, respectively. Values are represented as mean ± SEM (n = 6 rats/group). ##p < 0.01 versus the Control group; **p < 0.01, *p < 0.05 versus the HFD group.

Figure 3

Figure 3

YLZD upregulated the expression levels of hepatic SIRT1/Nrf2 pathway-related genes and proteins. (A) Typical IHC staining images of the liver with different treatments (magnification, ×200). (B,C) Average optical density values (AOD = IOD/Area) of SIRT1 and Nrf2 protein expression. (D) Expression of transcription factors associated with SIRT1/Nrf2 pathway were detected by Western blot. (E,F) Quantification of the corresponding Western blotting results in D. (G–J) RT-qPCR of relative mRNA expression of SIRT1, Nrf2, HO-1 and NQO1 in liver. Values are represented as mean ± SEM (n = 6 rats/group). ##p < 0.01, #p < 0.05 versus the Control group; **p < 0.01, *p < 0.05 versus the HFD group.

Figure 4

Figure 4

YLZD increased the concentration of serum SCFAs. (A) Serum total SCFAs, (B) serum AA, (C) serum PA, (D) serum BA, (E) serum IBA, (F) serum VA, (G) serum IVA, (H) serum HA, (I) serum IHA. AA, PA, BA, IBA, VA, IVA, HA and IHA are acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid and isohexanoic acid, respectively. Values are represented as mean ± SEM (n = 6 rats/group). ##p < 0.01, #p < 0.05 versus the Control group; **p < 0.01, *p < 0.05 versus the HFD group.

Figure 5

Figure 5

YLZD raised the concentration of fecal SCFAs. (A) Fecal total SCFAs, (B) fecal AA, (C) fecal PA, (D) fecal BA, (E) fecal IBA, (F) fecal VA, (G) fecal IVA, (H) fecal HA, (I) fecal IHA. Values are represented as mean ± SEM (n = 6 rats/group). ##p < 0.01, #p < 0.05 versus the Control group; **p < 0.01, *p < 0.05 versus the HFD group.

Figure 6

Figure 6

YLZD remodeled the composition of gut microbiota. (A,B) The bacterial diversity in intestinal estimated by alpha-diversity of chao and Shannon indexes. (C) Venn diagram of the overlap of the OTUs in the gut microbiota in different treatments. (D) The β-diversity analysis of Principal Coordinate Analysis (PCoA). (E) Bar plot of the gut microbiota at the phylum level. (F) Bar plot of the gut microbiota at the genus level. (G) The ratio of Firmicutes to Bacteroidetes. (H–M) Changes in the abundance of Christensenellaceae, Clostridia_UCG-014, Muribaculaceae, Prevotellaceae, Oribacterium and Lactobacillus, respectively. Values are represented as mean ± SEM (n = 6 rats/group). ##p < 0.01, #p < 0.05 versus the Control group; **p < 0.01, *p < 0.05 versus the HFD group.

Figure 7

Figure 7

Correlation between indicators of liver injury, oxidative stress-related enzymes, SCFAs levels and gut microbiota composition changes. Blue indicates positive correlation and red indicates negative correlation. **p < 0.01, *p < 0.05.

Figure 8

Figure 8

Effects of acetic acid and butyric acid on cell viability and FFA-induced lipid accumulation and oxidative stress in L02 cells. (A,B) The effects of AA and BA on L02 cell viability performed by CCK8 assays. (C) Cell TG level. (D) Cell Oil red O staining (magnification, ×400). (E) Quantitative analysis of oil red staining results was performed using Image Pro Plus 6.0 software. (F–H) The level of cell MDA, SOD and GSH-Px, respectively. L02 cells were pre-treated for 24 h with 1.0 mM FFA to constructed NAFLD cell model before treated with 1.0 mM AA or 0.5 mM BA for another 24 h. Data represent means ± SEM. ##p < 0.01 versus Control; *p < 0.05, **p < 0.01 versus FFA.

Figure 9

Figure 9

Butyric acid ameliorated FFA-induced L02 cells oxidative stress injury via the SIRT1/Nrf2 pathway. (A) Cell Oil red O staining (magnification, ×400). (B) Quantitative analysis of oil red staining results was performed using Image Pro Plus 6.0 software. (C) Cell TG level. (D–F) Western blot analysis of SIRT1 and Nrf2 in L02 cells. (G–I) The level of cell SOD, GSH-Px and MDA, respectively. (J,K) Histogram of intracellular ROS level detected by DCFH-DA. L02 cells were pre-treated for 24 h with 1.0 mM FFA to constructed NAFLD cell model before treated with 0.5 mM BA or 100 nM EX-527 for another 24 h. Data represent means ± SEM. ##p < 0.01 versus Control; *p < 0.05, **p < 0.01 versus FFA.

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