Protective Effects of Tiaoganquzhi Decoction in Treating inflammatory Injury of Nonalcoholic Fatty liver Disease by Promoting CGI-58 and Inhibiting Expression of NLRP3 Inflammasome

Abstract

Tiaoganquzhi Decoction (TGQZD) is a traditional Chinese herbal formulation demonstrated to be a clinically effective treatment for nonalcoholic fatty liver disease (NAFLD), although details concerning its clinical mechanism are poor. This study aimed to explore the mechanism of TGQZD on improvement of inflammatory damage and dyslipidemia caused by NAFLD through the CGI-58/ROS/NLRP3 inflammasome pathway. In our research, the in vivo protective effects of TGQZD on HFD-induced liver injury in rats and in vitro using lipopolysaccharide (LPS)+palmitate (PA)-stimulated HepG-2 cells model. Histological changes were evaluated by hematoxylin-eosin and Oil Red O staining. Inflammatory cytokines and protein expression were analyzed by ELISA, Real time PCR and western blotting. Liver function, blood lipids, free fatty acids (FFA), and reactive oxygen species (ROS) were determined by biochemical detection. Our results indicated that TGQZD exhibited anti-inflammatory activity, reduced the severity of NAFLD and ameliorated the pathological changes. Further, TGQZD improved liver function and lipid metabolism in NAFLD rats. TGQZD lowered serum aspartate aminotransferase, alanine aminotransferase, triglyceride, and total cholesterol levels. TGQZD suppressed the formulation of FFA and ROS. It also reduced the expression and release of the inflammatory cytokine interleukin-1β by promoting CGI-58 expression and inhibiting the expression of FFA, TNF-α, and the NLRP3 inflammasome induced by ROS. TGQZD exhibited anti-inflammatory effects via the CGI-58, ROS and NLRP3 inflammasome pathway in vivo and in vitro, respectively. Our findings demonstrated that TGQZD is a useful and effective therapeutic agent for treating NAFLD via promotion of CGI-58 to inhibit the expression of ROS-induced NLRP3 inflammasome.

Keywords: CGI-58; IL-1β; NLRP3; ROS; Tiaoganquzhi decoction; non-alcoholic fatty liver disease.

FIGURE 1

Identification of components of Tiaoganquzhi Decoction(TGQZD) using UPLC-MS/MS. The positive (A) and negative (B) ion chromatograms of TGQZD were shown as indicated. The major component and their retention time were listed in the (Table 2).

FIGURE 2

(A)Effect of TGQZD on liver histomorphology of NAFLD rats. The HE staining showed that the morphology and structure of the liver tissue in the ND group were normal; In the HFD group, fatty degeneration appeared in the liver. Hepatocytes are swollen and round and their volume is significantly higher than that of ND group. There exist a large number of fat vacuoles and balloons in cytoplasm. After the treatment of TGQZD, the fatty vacuoles and balloon like changes in NAFLD rats’ liver were improved. The results of oil red O staining showed that there was little red lipid drop in the liver tissue of the ND group and a large number of red lipid drops in the liver tissue of the HFD group, which showed that the lipid droplets infiltrated into the hepatocytes and fused into pieces. Red lipid droplets were also found in liver tissue of rats in TGQZD group, but the number and the fusion into pieces were less than that in HFD group (Figure 2). Histological examination showed that fatty degeneration, inflammation, and hepatocyte ballooning in TGQZD group were alleviated than HFD group (Table 3). (B) Effect of TGQZD on liver function and blood lipid of NAFLD rats. Compared with the ND group, the levels of ALT, AST, TG and TC in the HFD group were significantly higher (p < 0.05); compared with the HFD group, the levels of ALT, AST, TG and TC in the TGQZD group were significantly lower (p < 0.05). *p < vs. HFD Group, alone.

FIGURE 3

Effect of TGQZD on FFA and ROS content in liver of NAFLD rats. Compared with the ND group, the content of FFA and ROS in the liver of NAFLD rats was significantly increased (p < 0.05), while compared with the HFD group, TGQZD significantly reduced the level of FFA and ROS in the liver of NAFLD rats (p < 0.05). *p < 0.05 vs. HFD Group, alone.

FIGURE 4

Effect of TGQZD on serum IL-1 β content in NAFLD rats. After 8 weeks of treatment, compared with the ND group, the level of serum IL-1β in HFD group increased significantly (p < 0.05); while compared with the HFD group, the level of serum IL-1β in the TGQZD group decreased significantly (p < 0.05), indicating that TGQZD could effectively inhibit the formation of pro-inflammatory cytokines by reducing the increased level of IL-1 β in NAFLD rats. *p < 0.05 vs. HFD Group, alone.

FIGURE 5

Effects of TGQZD on the viability of HepG-2 cells. HepG-2 cells were treated with 10ug/ml LPS+0.4 mM palmitate and different concentrations of TGQZD for 24 h, and cell viability was measured using the MTT assay. Data are expressed as the mean ± SD (n = 3). *p < 0.05 vs. LPS + PA Group, alone.

FIGURE 6

Effect of water extract TGQZD on the formation of intracellular triglyceride (TG) and reactive oxygen species (ROS) in LPS + PA -treated HepG-2 cells. In the Control group, phosphate-buffered saline (PBS) in the culture medium; TGQZD group was treated with TGQZD in the culture medium; the LPS + PA group was treated with 10 μg/ml LPS + 0.4 mMPA in the culture medium and the LPS + PA + TGQZD group was treated with 4 mg/ml TGQZD and 10 μg/ml LPS + 0.4 mMPA in the culture medium, respectively. *p < 0.05 vs. LPS + PA Group, alone.

FIGURE 7

Effect of TGQZD on CGI-58 and NLRP3 inflammasome of HepG-2 cells. (A) Representative images of Western blot showing CGI-58 and NLRP3 inflammasome protein expression in HepG-2 cells in the different groups. (B) Quantitation of CGI-58 and NLRP3 inflammasome mRNA expression in HepG-2 cells in different groups. ACTIN was used as a loading control. *p < 0.05 vs. LPS + PA Group, alone, (n = 3).

FIGURE 8

Effect of TGQZD on CGI-58 and NLRP3 inflammasome of liver in NAFLD rats. (A) Representative images of Western blot showing CGI-58 and NLRP3 inflammasome protein expression in NAFLD rats in the different groups. (B) Quantitation of CGI-58 and NLRP3 inflammasome mRNA expression in NAFLD rats in different groups. ACTIN was used as a loading control. *p < 0.05 vs. HFD Group, alone, (n = 3).

FIGURE 9

The therapeutic effect of TGQZD on NAFLD rats. Based on the above, TGQZD can reduce the accumulation of lipid in liver by regulating the level of lipoproteins; improved hepatic function; inhibit FFA and promote CGI-58 expression to suppress activating the ROS-dependent NLRP3 inflammasome pathway, which reduce the liver inflammation injury.