Fig 2: Histological localization of NGF, proNGF and their cognate receptors, TrkA and p75NTR, in parenchymal hepatocytes of human livers with hepatolithiasis. Liver sections of lithiasis and contralateral parts in hepatolithiasis liver tissues (n=5) and paralesional sections of hepatocellular carcinoma livers (n=4) as non-hepatolithiasis control (NHC) underwent formalin-fixation, tissue-processing and paraffin-embedding procedures. Tissue sections were subjected to immunohistochemical staining for NGF, proNGF, TrkA and p75NTR. Scale bars=100 μm. Arrows indicate characteristic fatty changes in lithiasis tissues, while white and black arrowheads indicate immunoreactive signals in the hepatocytes and the infiltrated cells, respectively.

Fig 3: Expression of NGF, proNGF and their cognate receptors, TrkA and p75NTR, in human livers with hepatolithiasis. (a) Hepatolithiasis samples (n=5) included lithiasis and contralateral liver tissues. Paralesional protein extracts from patients with hepatocellular carcinoma were the non-hepatolithiasis controls (NHC, n=4) and were subjected to western blotting detection of NGF, proNGF, TrkA and p75NTR levels. The scan density data of NHC and hepatolithiasis groups are shown in Supplementary Tables S3 and S4, respectively. Densitometrical analyses of the expression levels of NGF and proNGF (b), as well as those of TrkA and p75NTR (c), are shown as the mean±s.e.m. of their ratios to internal actin controls. *P<0.05 vs NHC levels, #P<0.05 vs contralateral levels. (d) RT-qPCR detection of NGF, TrkA and p75NTR mRNA levels in human livers with hepatolithiasis. Spearman’s correlation analysis of the relationships of hepatic NGF (e) and proNGF expression (f) with serum total bilirubin levels.

Fig 4: Effects of TrkA and p75 NTR blocker treatments on bile duct ligation (BDL)-induced PARP cleavage and apoptosis of parenchymal hepatocytes. The mice receiving BDL surgery were intraperitoneally administered with either recombinant NGF (n=6) twice weekly or PBS (n=6), GW441756 (GW; n=6), DMSO (n=3) or PD90780 (PD; n=6) daily. After 2 weeks of treatment, pooled liver extracts of each group were subjected to western blotting to detect both propeptides and cleaved forms of caspase-3 and PARP (a). Density analysis of the activation of caspase-3 (b) and PARP (c), shown as the ratios of cleavage to propeptide content, followed by normalization to negative control (NC) levels. IHC staining for active PARP peptides (d) and labeling index (e) quantitatively demonstrated that PD90780 administration potentiated the increase of PARP activation in BDL-treated mouse livers. (f) Representative microphotographs of TUNEL staining showing the apoptotic events in mouse liver sections. Scale bars=50 μm. (g) Analysis of TUNEL-positive parenchymal cells indicated that recombinant NGF administration exerted a protective effect, whereas treatment with a p75NTR antagonist, PD90780, increased apoptotic cell death in BDL-induced cholestatic injured livers. All data are shown as the mean±s.e.m. *P<0.05 between indicated groups. ND, not detectable. NS, not significant.

Fig 5: Expression of SIRT1 in human hepatolithiasis and mouse cholestatic livers. (a) Western blotting detection of hepatic SIRT1 expression in human non-hepatolithiasis controls (NHC, n=4), as well as lithiasis and contralateral regions in human hepatolithiasis livers (n=5). NS, not significant. (b) RT-qPCR detection of SIRT1 mRNA in human hepatolithiasis livers. All data are shown as the mean±s.e.m. *P<0.05 compared with NHC. (c) Spearman’s correlation analysis of all of the human liver specimens suggested a positive correlation between hepatic SIRT1 and NGF/proNGF expression. (d) SIRT1 IHC staining showing both nuclear and cytoplasmic localization of SIRT1 peptides in hepatocytes of human NHC and hepatolithiasis livers. Bar=200 μm. (e) Upregulation of SIRT1 by NGF treatment and its abolishment by NGF-neutralizing antibodies in a mouse model of bile duct ligation (BDL) surgery-induced cholestatic liver injury. Mouse livers were collected 14 days post-BDL surgery. (f) SIRT1 IHC staining of mouse livers that received BDL surgery followed by NGF or saline administration for 14 days. Bar=100 μm.