Characterization of G4.5-COOH PAMAM Dendrimers
The FTIR spectrum of G4.5-COOCH3 and G4.5-COOH was shown in Fig. 1. Compared with G4.5-COOCH3, G4.5-COOH have a strong absorption band around 3430.37 cm− 1, corresponding to the stretching vibration peak of the hydroxyl group in the carboxylic acid. A strong carbonyl O = C-O characteristic absorption peak appeared near 1737.61 cm− 1, meanwhile a strong absorption peak of -C-O- appeared near 1414.87 cm− 1, indicating the presence of carboxyl groups (Fig. 1).
G4.5-COOH Alleviated ANIT-induced Cholestatic Liver injury in Mice
After gavage administration with ANIT, the mice presented mental sluggishness, messy and dull hair, dementia and physical inactivity, decreased appetite, reduced drinking, and dark yellow urine opposed to the normal group. The livers were dark red with white necrotic foci, the gallbladders were significantly enlarged with cholestasis, and the serum was dark and turbid. Report to ANIT group, -COOH + ANIT group showed significant improvement in spirit, hair, appetite and activity, the color of urine and liver was obviously lighter, the gallbladders were smaller and serum was clearer. However, after adding GW9662, the mice showed poorer spirit, hair, appetite and activity, darker urine, darker livers, larger gallbladders and more turbid serum than those in -COOH + ANIT group (Fig. 2).
Next, we detected the serum markers of liver functions including TBIL, DBIL, TBA, AST,ALT and ALP. Compared with control group, The levels of TBIL, DBIL, TBA, AST,ALT and ALP in ANIT group increased significantly( P < 0.01 or P < 0.001); Compared with ANIT group, the levels of TBIL, DBIL, TBA, AST,ALT and ALP in -COOH + ANIT group decreased significantly(P < 0.01 or P < 0.001); However, after adding GW9662, TBIL, DBIL, TBA, AST,ALT and ALP levels in GW9662 + -COOH + ANIT group were significantly higher than those in -COOH + ANIT group(P < 0.01 or P < 0.001)( Fig. 3).
G4.5-COOH Reduced Inflammation induced by ANIT treatment in Mice
The control group showed a normal portal triad comprising of the bile duct, hepatic artery and portal vein. In ANIT group, dilatation and hemorrhage of the portal vein, bile duct epithelial hyperplasia, bile duct stenosis and cholestasis were observed. Moreover, a large number of inflammatory cells infiltrated the portal area, with punctate and focal necrotic areas. In -COOH + ANIT group, the bile duct epithelium was slightly damaged and inflammatory cell infiltration was rare, almost returned to the conditions of the control group. In GW9662 + -COOH + ANIT group, bile duct epithelial hyperplasia, inflammatory cell infiltration and focal necrosis of the liver were increased(Fig. 4.A).
Immunohistochemical results demonstrated extensive macrophage infiltrated around the bile duct, vessels and in the necrotic lesions(P < 0.001).Compared with ANIT group, liver tissues from G4.5-COOH-treated mice demonstrated significant inhibition in macrophage and TNF-α infiltration(F4/80: P < 0.001,TNF-α༚P < 0.001). While macrophages and TNF-α significantly increased in GW9662 + -COOH + ANIT group༈F4/80༚P < 0.05. TNF-α༚P < 0.001༉(Fig. 4.B-E).
The expression of inflammatory factors were significantly increased in ANIT-induced mice compared with normal control mice (P < 0.001). The expression of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β was dramatically decreased in G4.5-COOH-treated mice(P < 0.001). However, in GW9662 + -COOH + ANIT group, the expression of inflammatory factors were higher than in -COOH + ANIT group(P < 0.01) (Fig. 4.F).
G4.5-COOH Decreased Cell Viability in A Concentration-Dependent Manner
In order to evaluate the cytotoxicity of G4.5-COOH and select appropriate concentration for further analysis, CCK8 was carried out. The results showed that G4.5-COOH decreased cell viability in a concentration-dependent manner. We chose the concentration of 7 uM which had little effect on cell viability for following experiments(Fig. 5).
G4.5-COOH Reduced OxS in Cholestatic Liver Injury
Compared with the control group, SOD and GSH levels in liver tissues of ANIT group were significantly lower ( P༜0. 001) while MDA level was significantly higher( P༜0. 001); Compared with ANIT group, the levels of SOD and GSH in -COOH + ANIT group increased significantly( P༜0. 001), while the level of MDA decreased significantly( P༜0. 001). However, after adding GW9662, SOD and GSH levels in GW9662 + -COOH + ANIT group were lower (P < 0.01 or P < 0.001)while MDA level was higher than those in the -COOH + ANIT group(P < 0.05)(Figure 6.A)
The same trend was observed in THLE cells. G4.5-COOH significantly reduced MDA level produced by TNF-α stimulation( P༜0. 001) and increased SOD and GSH levels(P < 0.01 or P < 0.001). However, this effect was weakened by GW9662(P < 0.05 or P < 0.01)༈Figure 6.B༉.
G4.5-COOH Reduced ER Stress in Cholestatic Liver Injury
The expressions of CHOP and GRP78 proteins in liver tissues of ANIT group were significantly higher than those of control group( CHOP:P༜0. 01,GRP78:P༜0. 001). Compared with the ANIT group, CHOP and GRP78 protein expressions in -COOH + ANIT group decreased obviously (CHOP:P༜0.05,GRP78:P༜0. 01). However, after adding GW9662, CHOP and GRP78 protein expressions in the GW9662 + -COOH + ANIT group were higher than those in -COOH + ANIT group(P < 0.05) (Fig. 7.A).
The same trend was observed in THLE cells. G4.5-COOH decreased CHOP and GRP78 protein expressions stimulated by TNF-α( CHOP:P༜0.05,GRP78:P༜0. 01), but this effect was weakened by GW9662(P < 0.01)(Fig. 7.B).
G4.5-COOH Reduced Liver Apoptosis in Cholestatic Liver Injury
The TUNEL staining and Bax、Bcl-2 protein results showed that compared with control group, the apoptosis rate was increased in ANIT group and reduced in -COOH + ANIT group(P < 0.05), indicating that G4.5-COOH inhibited liver cell apoptosis. However, after adding GW9662, the apoptosis rate was raised again(P < 0.05),indicating that GW9662 can antagonize the action of G4.5-COOH (Fig. 8.A-C).
Furthermore, THLE cells were incubated with Annexin V and propidium iodide (PI)dye and detected by a flow cytometer. The Flow Cytometry and Bax、Bcl-2 protein results showed that the apoptosis rate was significantly increased after interfering with TNF-α(P < 0.05), while G4.5-COOH reduced the rate of apoptosis and GW9662 increased the rate of apoptosis again༈P < 0.05༉.These results indicated that G4.5-COOH could inhibit THLE cell apoptosis which induced by TNF-α(Fig. 8.D-F).
G4.5-COOH increased PPAR-γ expression through inhibiting PI3K/ Akt/mTOR signaling pathway in Cholestatic Liver Injury
Next, we ought to investigate whether PI3K/Akt/mTOR signaling pathway was involved in the increase PPAR-γ induced by G4.5-COOH.The protein levels of PPAR-γ was down-regulated and p-Akt, p-mTOR were unregulated when exposing to ANIT or TNF-α(P < 0.05), and G4.5-COOH treatment further unregulated PPAR-γ expression and down-regulated p-Akt, p-mTOR expressions compared with ANIT or TNF-α group(P < 0.05), while the protein levels of Akt and mTOR were not significantly different between groups(P > 0.05). To further clarify the result, PPAR-γ inhibitor GW9662 was administrated to the mice and cells. Compared with G4.5-COOH + ANIT group, GW9662 markedly decreased the protein levels of PPAR-γ༈P < 0.05༉,demonstrating that the inhibitor was effective. However at the same time the phosphorylation of mTOR and Akt was up-regulated again༈P < 0.05༉. (Fig. 9.A-D)