Plumbagin protects liver against fulminant hepatic failure and chronic liver fibrosis via inhibiting inflammation and collagen production

Abstract

Plumbagin is a quinonoid constituent extracted from Plumbago genus, and it exhibits diverse pharmacological effects. This study thoroughly investigated the effects of plumbagin on thioacetamide-induced acute and chronic liver injury. Results shown that plumbagin increased survival rate, reduced liver congestion and inflammation, and decreased macrophages and neutrophils in the fulminant hepatic failure model, and remarkably diminished liver fibrosis and inflammation in the chronic liver injury model. Furthermore, plumbagin significantly suppress the HSCs/myofibroblasts activation by reduced expression of markers α-SMA and COL-1/3, and reduced macrophage in liver. In the in vitro study, plumbagin induced apoptosis and suppressed the proliferation of LX-2 cells (human HSCs). Plumbagin treatment increased AMPK phosphorylation and attenuated NF-κB, STAT3, and Akt/mTOR signals in LX-2 cells, while SMAD2 phosphorylation was not changed. Noticeably, plumbagin promoted AMPK binding to p300 which is a cofactor of SMAD complex, this may further competitively decreases the p300/SMAD complex initiated transcription of COL-1/3 and α-SMA. Additionally, plumbagin hampered inflammation related NF-κB signal in RAW 264.7 cells. In conclusion, these findings indicate that plumbagin may be a powerful drug candidate to protect the liver from acute and chronic damage by inhibiting inflammation and collagen production.

Keywords: fulminant hepatic failure; hepatic stellate cell; inflammation; liver fibrosis; plumbagin.

Figure 1. Plumbagin protected mice from fulminant hepatic failure

A. Survival rates of PL+TAA and TAA mice. Acute liver injury was established using a single injection of 4% TAA (300 μg/g bw ip). The presence of plumbagin (2 μg/g bw ig) effectively reduced TAA-induced death. B. The level of ALP/ALT 96 h after intraperitoneal TAA infusions. C. Gross histology of livers. Acute liver injury was induced, and mice were sacrificed at the progressive daily points. Left: Gross liver histology. Right: Enlargement of liver tissues (in red circle of the left). The presence of plumbagin significantly diminished the congestion (arrows denote) due to TAA-induced hemorrhage. Representative images are shown for all panels. Mean ± SD, ns (not significant), * p<0.05, **p<0.01 were calculated using two-tailed Student's t test (T TEST).

Figure 2. Plumbagin decreased congestion and inflammatory cell infiltration and macrophage recruitment in the FHF model

A. Congestion (cyan arrows) and inflammatory cell infiltration (yellow arrows) was assayed using HE staining. B. Necroinflammatory scores were determined using the Ishak classification. C. Immunostaining of F4/80 (black arrows). D. Positive cells of F4/80 were assessed by manual method. E. Immunostaining of MPO. Left panel, pictures; right panel, quantification of left images. F. Hepatic content of MCP-1. Representative images are shown for all panels. ns (not significant), * p<0.05, **p< 0.01; ***p< 0.001. Data are expressed as means ± SD. Statistical analyses were performed using Student's t test.

Figure 3. Plumbagin suppressed TAA-induced collagen deposition, inflammatory cell infiltration, and liver function abnormalities in a chronic liver damage model

A. Fibrosis (green arrows) was determined using sirius red staining. B. Infiltration of immune cells (yellow arrows) was shown using HE. C. F4/80 protein (black arrows) was measured using immunohistochemical staining (brown, HRP-conjugated and developed using DAB). Sections were counterstained with hematoxylin (dark blue). D. ALP and ALT levels. E. Periodic acid Schiff staining for hepatocellular glycogen storage (glycogen in red). Patches of periodic acid Schiff negative, nonfunctional hepatocytes were shown by blue arrows. Histopathological evaluations were conducted using Ishak's scoring system and Image-Pro Plus 6 Windows Software. Representative images are shown for all panels. Data are expressed as means ± SD. ns (not significant), *p< 0.05; **p< 0.01. p values were calculated using two-sided Student's t tests.

Figure 4. Immunofluorescence assays of HSCs/myofibroblasts

A. α-SMA, a marker of activated HSCs/myofibroblasts; B. COL-1 and C. COL-3, markers of ECM produced by HSCs/myofibroblasts; D. TGF-β1. The TGF-β1 signal is involved in HSCs/myofibroblasts. HRP-(brown) or Alexa 555 (red)-conjugated secondary antibodies were used. The sections were counterstained with hematoxylin (dark blue) or DAPI (blue). Representative images are shown for all panels. The column charts given in figure are means ± SD values from three separate experiments.*p< 0.05; **p< 0.01. Statistical analyses were performed using Student's t test.

Figure 5. Plumbagin enhanced hepatocellular proliferation and inhibited hepatocellular apoptosis in a chronic liver fibrosis model

A. Ki67 (yellow arrows), a marker of proliferation. B. Cleaved caspase-3, a marker of apoptosis (black arrows). The sections were incubated with HRP-conjugated secondary antibodies and developed using DAB (brown). The sections were counterstained with hematoxylin (dark blue). Data are expressed as means ± SD, and data are representative of 4-6 mice/group. *p< 0.05; **p< 0.01. Statistical analyses were performed using Student's t tests.

Figure 6. Effects of plumbagin on human hepatic stellate cells

A. Plumbagin promoted pAMPK levels in a time-dependent manner. LX-2 cells were treated with 10 μM plumbagin for the indicated times, and pAMPK levels were analyzed. B. Plumbagin inhibited pSTAT3, p-p65, pAkt and p-mTOR levels in a dose-dependent manner. However, plumbagin had no effect on pSMAD2. LX-2 cells were first treated with the indicated concentrations of plumbagin for 1 h after incubation. Whole cells were harvested and analyzed using Western blots for pAMPK, pSTAT3, p-p65, pAkt, p-mTOR and pSMAD2. GAPDH was used as a loading control. C. Plumbagin facilitated the bind of AMPK with p300. LX-2 cells were treated with 10 μM plumbagin for 1 h, and cell lysates were immunoprecipitated using an anti-AMPK antibody, followed by immunoblotting with an anti-p300 or anti-AMPK antibody. D. Plumbagin decreased mRNA level of fibrosis-associated genes. LX-2 cells were treated with plumbagin (200 ng/ml) or TGF-β1 (2 ng/ml) or combination for 24 h, and then total RNA were extracted for quantitative PCR of COL-1, COL-3 and α-SMA. Representative images are shown for all panels. Data are expressed as means ± SD. *p< 0.05; **p< 0.01. p values were calculated using a two-sided Student's t test.

Figure 7. Liver protection mechanism of plumbagin

Plumbagin increased AMPK phosphorylation that promoted AMPK binding to p300, which is a SMAD transcriptional cofactor. This may further competitively decreases the p300/SMAD complex initiated transcription for COL-1/3 and α-SMA. Plumbagin also attenuated NF-κB, STAT3, and Akt/mTOR signals in LX-2 cells, which were involved in pro-inflammation and survival of HSCs/myofibroblasts. In conclusion, these findings indicate that plumbagin may be a powerful drug candidate to protect the liver from acute and chronic damage by inhibiting inflammation and collagen production.