ABSTRACT
Objective:To evaluate the effects of berberine on necroptosis of non-alcoholic fatty liver disease in mice and its relationship with adenosine monophosphate-activated protein kinase(AMPK)/ signal transducer and activator of transcription 6(STAT6) pathway.Methods:Twenty-five 8-week-old male C57BL/6N mice were divided into control group, steatotic liver group, berberine treatment group(200 mg·kg -1·d -1), AMPK inhibitor Compound C treatment group(0.2 mg·kg -1·d -1), and STAT6 inhibitor AS1517499 treatment group(10 mg·kg -1·d -1). After 12 weeks of intervention, the mice and liver tissue were weighed, and serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), triglyceride, tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) as well as liver malondialdehyde and superoxide dismutase were measured; liver tissue HE, Masson, and oil red O staining were performed. Western blotting was used to detect the expressions of necroptosis related proteins[receptor interaction protein kinase 3(RIPK3), phosphorylated(p-) mixed lineage kinase domain-like(MLKL)], AMPK, p-AMPK, and p-STAT6. Results:Compared with control group, the steatotic liver group had higher quality of liver and liver index, and higher levels of serum AST, ALT, triglyceride, TNF-α, IL-1β, and oxidative stress( P<0.05); Liver tissue was full of cavity changes and inflammatory cell infiltration, widely distributed red lipid droplets and obvious blue fiber dyeing; The expressions of RIPK3 and p-MLKL were up-regulated ( P<0.05), but the levels of p-AMPK and p-STAT6 were relatively reduced ( P<0.05). Compared with the steatotic liver group, berberine intervention decreased liver quality and liver index, improved liver function, reduced blood lipid levels, pro-inflammatory factor expression and oxidative stress level, and significantly alleviated the degree of liver steatosis and fibrosis, the levels of RIPK3 and p-MLKL ( P<0.05), while the expressions of p-AMPK and p-STAT6 were increased significantly ( P<0.05). As compared with the berberine treatment, AMPK and STAT6 inhibitor treatment could offset the protective effect of berberine on steatotic liver, moreover, the expressions of RIPK3 and p-MLKL were increased ( P<0.05). There was no statistical difference in AMPK total protein content among the five groups ( P>0.05). Conclusion:Berberine can activate AMPK/STAT6 pathway to inhibit the necroptosis of hepatocyte, thus plays a protective role on non-alcoholic fatty liver disease in mice.
ABSTRACT
Objective To evaluate the role of silent information regulator fac tor 2-related enzyme 1 (SIRT1)/Forkhead Box O3 (FoxO3a) signaling pathway in berberine pretreatment-induced reduction of hypoxia/reoxygenation (H/R)-caused injury to hepatic parenchymnal cells.Methods Hepatic parenchymal cells obtained from AML12 mice were cultured and seeded in 6-well plates (2 ml/well) and in 96-well plates (200 μl/well) at the density of l×l06 cells/ml.The cells were divided into 4 groups (n=36 each)using a randomn number table:control group (group C),group H/R,berberine pretreatment group (group BP) and SIRT1-siRNA group (group SS).The cells were cultured in normal culture atmosphere (5％ CO2-21％ O2-74％ N2) in group C.In H/R,BP and SS groups,the cells were exposed to hypoxic air (5％ CO2-1％ O2-94％ N2) for 12 h,followed by 6 h reoxygenation in normal culture atmosphere (5％ CO2-21％ O2-74％ N2).In group SS,small interference RNA targeting SIRT1 (SIRT1-siRNA) was added to the culture medium at 24 h prior to hypoxia.Berberine (final concentration 5 μmol/L) was added at 2 h prior to hypoxia in BP and SS groups.At the end of reoxygenation,the cell viability was measured by methyl thiazolyl tetrazolium assay,the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined using enzyme-linked immunosorbent assay,cell apoptosis was detected by flow cytometry,the expression of SIRT1 and FoxO3α was detected by Western blot,and the acetylation of FoxO3α was measnred by using immunoprecipitation.Apoptotic rate was calculated.Results Compared with group C,the cell viability was significantly decreased,the MDA content was increased,the SOD activity was decreased,apoptotic rate was increased,the expression of SIRT1 and ratio of FoxO3α expression in nucleus/in cytoplasma were increased,and the acetylation of FoxO3α in the nucleus was increased in H/ R,BP and SS groups (P＜ 0.05).Compared with group H/R,the cell viability was significantly increased,the MDA content was decreased,the SOD activity was increased,apoptotic rate was decreased,the expression of SIRT1 and ratio of FoxO3α expression in nucleus/in cytoplasma were increased,and the acetylation of FoxO3α in the nucleus was increased in group BP (P＜0.05).Compared with group BP,the cell viability was significantly decreased,the MDA content was increased,the SOD activity was decreased,apoptotic rate was increased,the expression of SIRT1 and ratio of FoxO3α expression in nucleus/in cytoplasma were decreased,and the acetylation of FoxO3α in the nucleus was decreased in group SS (P＜O.05).Conclusion The mechanism by which berberine pretreatment attenuates H/R-caused injury to hepatic parenchymal cells is related to promotion of SIRT1 expression in cells and inhibition of FoxO3α acetylation in the nucleus.