Erhong Jiangzhi Decoction Inhibits Lipid Accumulation and Alleviates Nonalcoholic Fatty Liver Disease with Nrf2 Restoration Under Obesity

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) refers to the liver pathological changes caused by excessive fat accumulation in hepatocytes owing to various reasons, which has become an emerging health challenge. Erhong Jiangzhi Decoction (EHJD) is a traditional Chinese medicine decoction. This study aims to investigate the therapeutic effect of EHJD on NAFLD.

Methods: NAFLD model was constructed by high-fat diet (HFD)-induced mice and oleic acid-induced HepG2 cells. Mice were intragastrically administered with EHJD and HepG2 cells were treated with EHJD drug-containing serum. The effects of EHJD on NAFLD were explored in vivo and in vitro. Histological assessment was performed by hematoxylin-eosin and oil red O staining. ELISA was exploited to detect the expression of lipid accumulation, liver function, inflammation, and oxidative stress related indicators. The expression of Nrf2/HO-1 pathway was detected by qRT-PCR and Western blot.

Results: In HFD-induced NAFLD mice, the body weight was increased, and liver/weight, inguinal fat/weight, and epididymal fat/weight were higher, while EHJD reduced them. Staining results exhibited that EHJD decreased inflammatory cell infiltration and oil red lipid droplets in HFD-induced mice. In addition, EHJD treatment suppressed TC, TG, ALT and AST levels; TNF-α, IL-1β, IL-6 and MDA levels were inhibited by EHJD, while GSH-Px, CAT and T-AOC levels were increased in NAFLD through the in vivo and in vitro experiments. The suppression of Nrf2 weakened the inhibitory effect of EHJD on lipid metabolism, liver injury, inflammation and oxidative stress.

Conclusion: EHJD had a protective effect on NAFLD by alleviating lipid accumulation and liver injury, inhibiting inflammation, and oxidative stress, which was achieved by the restoration of Nrf2.

Keywords: Erhong Jiangzhi Decoction; Nrf2/HO-1 signaling pathway; lipid accumulation; nonalcoholic fatty liver disease; oxidative stress.

Figure 1

Effects of Erhong Jiangzhi Decoction (EHJD) on body weight, related organ indexes and liver histopathology of NAFLD model mice induced by high-fat diet (HFD) (n=6). (A) Changes in body weight of mice. **P<0.01 vs 0d. (B–E) Organ (liver, heart) and adipose tissue (epididymal fat, inguinal fat) weight ratio. (F) Hematoxylin-eosin staining of liver tissues (× 200, 100 μm). (G). Oil red O staining of liver tissues (× 200, 100 μm). Black arrows indicate oil-red droplets. **P<0.01 vs Control ^#P<0.05 ^## P<0.01 vs NAFLD.

Figure 2

Erhong Jiangzhi Decoction (EHJD) improved lipid accumulation and liver injury in NAFLD model mice induced by high-fat diet (HFD) (n=6). (A) ELISA detection of blood lipid markers (TC, TG, LDL-C, and HDL-C). (B) ELISA detection of liver function markers (ALT, AST). *P<0.05 **P<0.01 vs Control ^#P<0.05 ^## P<0.01 vs NAFLD.

Figure 3

Erhong Jiangzhi Decoction (EHJD) inhibited liver inflammation and oxidative stress in NAFLD model mice induced by high-fat diet (HFD) (n=6). (A) ELISA detection of the levels of TNF-α, IL-1β, and IL-6. (B) ELISA detection of the levels of antioxidant markers (MDA, GSH-Px, CAT, and T-AOC). *P<0.05 **P<0.01 vs Control ^#P<0.05 ^## P<0.01 vs NAFLD.

Figure 4

Erhong Jiangzhi Decoction (EHJD) regulated Nrf2/HO-1 signaling pathway in NAFLD model mice induced by high-fat diet (HFD). (A) RT-qPCR detection of the expression of Nrf2 and HO-1 (n=6). (B) Western blot detection of protein expression of Nrf2 and HO-1 (n=3). **P<0.01 vs Control ^#P<0.05 ^## P<0.01 vs NAFLD.

Figure 5

Effects of Erhong Jiangzhi Decoction (EHJD) drug containing serum on viability and lipid accumulation in HepG2 cells induced by oleic acid (OA). (A) CCK-8 assay was performed to detect cell viability. (B) Oil red O staining of HepG2 cells (×200, 100 μm). (C) ELISA was used to detect the levels of lipid markers (TC, TG). (D) ELISA was used to detect the levels of liver function markers (ALT, AST) (n=6). **P<0.01 vs Control ^## P<0.01 vs OA.

Figure 6

Effects of Erhong Jiangzhi Decoction (EHJD) drug containing serum on inflammatory factors and antioxidant markers in HepG2 cells induced by oleic acid (OA) (n=6). (A) ELISA was used to detect the levels of inflammatory factors (TNF-α, IL-1β and IL-6). (B) ELISA was used to detect the levels of antioxidant markers (MDA, GSH-Px, CAT, and T-AOC). **P<0.01 vs Control ^#P<0.05 ^## P<0.01 vs OA.

Figure 7

Erhong Jiangzhi Decoction (EHJD) drug containing serum regulated Nrf2/HO-1 signaling pathway in HepG2 cells induced by oleic acid (OA) (n=3). **P<0.01 vs Control ^#P<0.05 ^## P<0.01 vs OA.

Figure 8

EHJD drug containing serum alleviated lipid accumulation and liver function injury in oleic acid (OA)-induced HepG2 cells through Nrf2/HO-1 pathway. (A) RT-qPCR was performed to verify the silencing efficiency of Nrf2/HO-1 pathway (n=6). **P<0.01 vs Control. (B) Western blot was used to detect the expression of Nrf2 and HO-1 (n=3). (C) Oil red O staining results of OA-induced HepG2 cells (×200, 100 μm). (D–E). ELISA was executed to measure the levels of blood lipid markers (TC, TG) and liver function markers (ALT, AST) (n=6). **P<0.01 vs OA ^##P<0.01 vs si-NC.

Figure 9

EHJD drug containing serum inhibited inflammation and oxidative stress in oleic acid (OA)-induced HepG2 cells through Nrf2/HO-1 pathway. (A and B) ELISA was executed to measure the levels of inflammatory factors (TNF-α, IL-1β and IL-6) and antioxidant markers (MDA, GSH-Px, CAT, and T-AOC) (n=6). **P<0.01 vs OA ^##P<0.01 vs si-NC.

Figure 10

The mechanism diagram of Erhong Jiangzhi Decoction (EHJD) on nonalcoholic fatty liver disease (NAFLD).