| Acute liver injury(ALI)is a serious disease in which large area of liver cell necrosis or apoptosis,hepatocyte steatosis,inflammatory reaction,oxidative stress and liver function damage are caused by various factors in a short period of time,usually with poor prognosis and high mortality rate.Inflammation and oxidative stress are the key factors in the pathogenesis of ALI.Due to the rapid course of ALI and the lack of effective therapeutic drugs,it is urgent to develop new drugs.Pterostilbene(Pte),a natural polyphenol product extracted from blueberries and grapes,has been reported that exerted multiple biological activities,including antioxidative,anti-inflammatory,anti-carcinogenic,and anti-apoptotic properties.As a classic endotoxin,lipopolysaccharide(LPS)can trigger the activation of immune cells and trigger macrophage release inflammatory cytokines and initiate the inflammatory response through multiple signaling pathways.D-galactosamine(D-Gal)is one of the hepatotoxic substances that inhibits the synthesis of RNA and protein in hepatocytes,caused metabolic disorders of sugars and phospholipids,and finally sensitizes the lethality of LPS.LPS combined with D-Gal can cause diffuse liver necrosis and inflammation.The pathological characteristics of ALI model in mice established by LPS/ D-Gal were similar to those of clinical viral hepatitis,and it has been widely used to explore the mechanism of liver injury and potential therapeutic drugs.However,there is very little data showing the hepatoprotective effect of Pte on LPS/D-Gal induced ALI in mice.In this study,ALI model induced by LPS/D-Gal is established to investigate the possible hepatoprotective effect and underlying mechanism of Pte,and further validated on mouse AML12 cells.The main research content and results of this paper are as follows:Male BALB/c mice(6–8-week-old;weight 18–22 g)are randomly divided into six groups as the following:(1)control group;(2)LPS/D-Gal group(LPS 50μg/kg and D-Gal 500 mg/kg);(3–5)LPS/D-Gal + Pte(10,20,and 40 mg/kg)groups and(6)Pte(40 mg/kg)only group.Prior to the LPS/D-Gal challenge,Pte(10,20,and 40 mg/kg)is injected intraperitoneally twice in groups 3 to 6(12 h apart).One hour after the last pretreatment of Pte,mice in 2 to 5 groups are treated intraperitoneally with LPS(50 μg/kg)and D-Gal(500 mg/kg)to establish ALI models.All animals are sacrificed 9 h after LPS/D-Gal treatment to obtain blood samples and liver tissues.HE staining,Western blotting,q RT-PCR,ELISA were used to detect the changes of blood biochemical indexes,histopathology,liver antioxidant level,inflammatory cytokine secretion,NF-κB and Nrf2 /HO-1signaling pathway key protein phosphorylation levels.It has been found that Pte markedly ameliorates LPS/D-Gal-induced inflammatory infiltration,hemorrhage,and dissociation of the hepatic cord,reducing the myeloperoxidase(MPO)activity in liver tissues and serum levels of alanine transaminase(ALT)and aspartate aminotransferase(AST)in ALI.Pte also inhibits LPS/D-Gal-induced secretion of pro-inflammatory cytokine tumor necrosis factor-a(TNF-α),interleukin 6(IL-6),and interleukin 1β(IL-1β)in liver tissues,and the production of MDA and consumption of SOD enzyme were significantly reduced.Furthermore,the western blot analysis reveals that LPS/D-Gal-activated NF-κB is significantly inhibited by Pte,and Nrf2 and HO-1 are upregulated by Pte.Secondly,mice normal hepatocytes(AML12)were used to verify the liver protective mechanism of Pte in vitro.CCK-8 was used to test the toxic effect of Pte on AML12,and to determine the safe dose range of Pte.The AML12 were divided into blank group,LPS group,LPS+ Pte(5μM)group,LPS+ Pte(10μM)group,LPS+ Pte(20μM)group,and seeded into 24-well plate and 6-well plate respectively.After pretreatment with Pte for 1h,LPS(final concentration 1μg/m L)was added to stimulate 6h,and the same amount of medium was added to the blank group.Supernatant and cell proteins were collected for measurement.It has been found that the activity of hepatocytes of AML12 was not affected by the treatment of Pte at the concentration of 2.5 to 20μM for 12 h.Pte inhibits LPS-induced secretion of pro-inflammatory cytokine TNF-α,IL-6 and IL-1β into the supernatant.Furthermore,the western blot analysis reveals that LPS-activated NF-κB is significantly inhibited by Pte,and Nrf2 and HO-1 are upregulated by Pte.To sum up,this study established LPS/D-Gal-induced ALI and LPS-induced AML12 cell inflammation model,combined in vivo and in vitro experiments to preliminary explore the effect of Pte on LPS/D-Gal-induced ALI.The results showed that Pte could reduce the inflammatory response of liver and AML12 cells by inhibiting the activated NF-κB signaling pathway,and enhance the antioxidant capacity by activating Nrf2/HO-1 signaling pathway.This study combined in vivo and in vitro experiments to explore the protective effect and mechanism of Pte on LPS/ D-Gal-induced acute liver injury in mice,providing preliminary basis and reference for the application of Pte as a liver protective additive. |
