Huang Gan Formula Alleviates Systemic Inflammation and Uremia in Adenine-Induced Chronic Kidney Disease Rats May Associate with Modification of Gut Microbiota and Colonic Microenvironment

Abstract

Purpose: This study aims to investigate the effects of Huang Gan formula (HGF), a Chinese herbal prescription used for chronic kidney disease (CKD), on the regulation of the gut microbiota and colonic microenvironment of CKD.

Methods: CKD rats were induced by 150 mg/kg adenine gavage for 4 weeks, then orally treated with or without 3.6 g/kg or 7.2 g/kg of HGF for 8 weeks. The renal function and structure were analyzed by biochemical detection, hematoxylin and eosin, Masson's trichrome, Sirius red and immunochemical staining. Average fecal weight and number in the colon were recorded to assess colonic motility. Further, the changes in the gut microbiota and colonic microenvironment were evaluated by 16S rRNA sequencing, RT-PCR or immunofluorescence. The levels of inflammatory cytokines, uremic toxins, and NF-κB signaling pathway were detected by RT-PCR, ELISA, chloramine-T method or Western blotting. Redundancy analysis biplot and Spearman's rank correlation coefficient were used for correlation analysis.

Results: HGF significantly improved renal function and pathological injuries of CKD. HGF could improve gut microbial dysbiosis, protect colonic barrier and promote motility of colonic lumens. Further, HGF inhibited systemic inflammation through a reduction of TNF-α, IL-6, IL-1β, TGF-β1, and a suppression of NF-κB signaling pathway. The serum levels of the selected uremic toxins were also reduced by HGF treatment. Spearman correlation analysis suggested that high-dose HGF inhibited the overgrowth of bacteria that were positively correlated with inflammatory factors (eg, TNF-α) and uremic toxins (eg, indoxyl sulfate), whereas it promoted the proliferation of bacteria belonging to beneficial microbial groups and was positively correlated with the level of IL-10.

Conclusion: Our results suggest that HGF can improve adenine-induced CKD via suppressing systemic inflammation and uremia, which may associate with the regulations of the gut microbiota and colonic microenvironment.

Keywords: chronic kidney disease; colonic microenvironment; gut-kidney axis; systemic inflammation.

Figure 1

HGF ameliorates renal structure and function of rats with CKD. (A) Schematic diagram of the experiments. (B) Representative pictures of the kidneys. (C) The left and right kidney indexes. n = 7–10 rats per group. (D and E) Representative photomicrographs of H&E, Masson, and Sirius Red stains (400×) (D), and areas of collagen fiber quantified by ImageJ software (E). n = 7–9 rats per group. (F and G) Immunochemistry of fibronectin and type I collagen in the renal tissues (400×) (F), and positive expressions were quantified by ImageJ software (G). n= 7–9 rats per group. Data are presented as mean ± SD. *P < 0.05 and ***P < 0.001.

Figure 2

HGF improves the gut microbial dysbiosis in adenine-induced CKD. (A–D) In the 4th week, alpha diversity (A), beta diversity (B), relative abundance at the phylum level (C) and Firmicutes/Bacteroidetes (F/B) ratio (D) of the gut microbiota were analyzed. (E–H) In the 8th week, alpha diversity (E), beta diversity (F), relative abundance at the phylum level (G) and F/B ratio (H) of the gut microbiota were analyzed. (I and J) Relative abundance of differential bacteria between CKD and CKD + HGF-H group in the 4th (I) and 8th (J) weeks are shown with Wilcoxon rank-sum test bar plot. Red asterisk (*, **, ***) in (I and J) indicate P < 0.05, 0.01 or 0.001, respectively. n = 6–8 rats per group (4th week), and n = 5 rats per group (8th week). Data represent mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

Effects of HGF on the colonic microenvironment in the CKD rats. (A and B) Fecal shape (A), and weight per pellet (B) that was calculated as (total wet weight) / (total numbers), n = 7–8 rats per group. (C and D) Number of residual stools in the colon, n = 7–8 rats per group. (E) Representative images of the colonic tissues using H&E stains (200×). The black arrows indicate injuries. (F) mRNA expression of ZO-1 and Occludin in colon tissues, n = 3 from three independent experiments. (G and H) Representative pictures of expression of ZO-1 and Occludin in colon from immunofluorescence staining (400×). (I) The mean fluorescence intensity of ZO-1 and occludin was quantified by ImageJ software, n = 6 rats per group. Data represent mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4

HGF suppresses blood’s inflammation of the CKD rats. Concentration of TNF-α (A), IL-6 (B), IL-1β (C) and IL-10 (D) in rat serum were determined by ELISA, n = 7–8 rats per group. Data represent mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

HGF alleviates renal inflammation by inhibiting NF-κB signaling pathway. (A–C) The mRNA expression of TNF-α (A), IL-6 (B) and TGF-β1 (C) in renal tissue were detected by RT-PCR. (D and E) The protein expression of TNF-α, IL-6, TGF-β1, NF-κB, p-NF-κB, IκBα, and p-IκBα in renal tissues were detected by Western blot. (F–H) Optical density of TNF-α, IL-6 and TGF-β1 (F), NF-κB and p-NF-κB (G), IκBα and p-IκBα (H) was quantified by ImageJ software, n = 3 rats per group. The visualization of the bands was performed by using Rapid Auto-exposure mode of the Image Lab^TM Touch Software that supported by ChemiDoc^TM System from the Bio-Rad. The exposure time for GAPDH was 3 to 10 second, while that for the tested proteins were a few seconds to several minutes. All data represent mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6

HGF reduces the level of uremic toxins in CKD. The concentration of indoxyl sulfate (IS) (A), p-cresol sulfate (PCS) (B), advanced oxidation products (AOPPs) (C), and endotoxin (D) were detected by ELISA, n = 5–6 rats per group. Data represent mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 7

HGF suppresses systemic inflammation and uremia by regulating the intestinal flora. (A) RDA plots show relationship between the selected cytokines. (B) The bacteria enriched in groups was determined using linear discriminant analysis. (C) Spearman correlation heatmap presents correlations between the gut flora, inflammatory cytokines and uremic toxins. P-values of the correlation were shown: *P < 0.05, **P < 0.01, and ***P < 0.001.