Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

Abstract

Aim: To investigate the potential mechanism of Arg-Gly-Asp (RGD) peptide-labeled liposome loading oxymatrine (OM) therapy in CCl₄-induced hepatic fibrosis in rats.

Methods: We constructed a rat model of CCl₄-induced hepatic fibrosis and treated the rats with different formulations of OM. To evaluate the antifibrotic effect of OM, we detected levels of alkaline phosphatase, hepatic histopathology (hematoxylin and eosin stain and Masson staining) and fibrosis-related gene expression of matrix metallopeptidase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-1 as well as type I procollagen via quantitative real-time polymerase chain reaction. To detect cell viability and apoptosis of hepatic stellate cells (HSCs), we performed 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay and flow cytometry. To reinforce the combination of oxymatrine with HSCs, we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM, and its targeting of HSCs was examined by fluorescent microscopy.

Results: OM attenuated CCl₄-induced hepatic fibrosis, as defined by reducing serum alkaline phosphatase (344.47 ± 27.52 U/L vs 550.69 ± 43.78 U/L, P < 0.05), attenuating liver injury and improving collagen deposits (2.36% ± 0.09% vs 7.70% ± 0.60%, P < 0.05) and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen (P < 0.05). OM inhibited cell viability and induced apoptosis of HSCs in vitro. RGD promoted OM targeting of HSCs and enhanced the therapeutic effect of OM in terms of serum alkaline phosphatase (272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L, P < 0.05), liver injury, collagen deposits (0.26% ± 0.09% vs 2.36% ± 0.09%, P < 0.05) and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen (P < 0.05). Moreover, in vitro assay demonstrated that RGD enhanced the effect of OM on HSC viability and apoptosis.

Conclusion: OM attenuated hepatic fibrosis by inhibiting viability and inducing apoptosis of HSCs. The RGD-labeled formulation enhanced the targeting efficiency for HSCs and the therapeutic effect.

Keywords: Arg-Gly-Asp peptide; Hepatic fibrosis; Hepatic stellate cell; Oxymatrine; Target therapy.

Figure 1

Oxymatrine liposomes attenuated hepatic fibrosis and improved collagen deposition. A: Representative images of liver treated with oxymatrine (OM) in different liposomal formulations in rats with CCl₄-induced hepatic fibrosis. Liver tissues were obtained at 4 wk after treatment and stained with HE; B: Representative histological images of liver treated with OM in different liposomal formulations in rats with CCl₄-induced hepatic fibrosis. Liver tissues were obtained at 4 wk after treatment and stained with Masson stain (original magnification × 100). HE: Hematoxylin and eosin.

Figure 2

Oxymatrine liposomes induced apoptosis in hepatic stellate cells in vitro. A: Inhibitory effect of oxymatrine (OM) on hepatic stellate cell (HSC) viability in vitro. HSCs were isolated and treated with OM in different liposomal formulations. Cell viability was determined using MTT assay; B: Electron micrograph of untreated HSCs demonstrates the normal structure of HSCs. OM-liposome-treated (24 h) HSCs had morphological features of apoptosis: cell shrinkage and apoptotic body formation. OM-RGD-liposome-treated HSCs showed typical morphological features of apoptosis: cell shrinkage and apoptotic body formation. RGD-liposome-treated HSCs showed normal structure; C: Cell cycle analysis after induction of apoptosis in HSCs by OM via flow cytometry. The cells were incubated with different formulation of OM for 24 h, and stained with PI. MTT: 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide; RGD: Arg-Gly-Asp; PI: Propidium iodide; DI: DNA grading index.

Figure 3

Oxymatrine liposomes inhibited fibrosis-related gene expression. The expression of fibrosis-related gene, such as MMP-2, TIMP-1 and collagen I was evaluated by real-time polymerase chain reaction. ^aP < 0.05 vs normal; ^cP < 0.05 vs CCl₄-induced heaptic fibrosis; ^eP < 0.05 vs oxymatrine liposomes. MMP: Matrix metallopeptidase; TIMP: Tissue inhibitor of metalloproteinase; OM: Oxymatrine; RGD: Arg-Gly-Asp.

Figure 4

Representative fluorescein isothiocyanate hepatic stellate cells in rat hepatic tissue treated with oxymatrine liposomes and oxymatrine-Arg-Gly-Asp liposomes (original magnification × 200). OM: Oxymatrine; RGD: Arg-Gly-Asp.