Protective effect of clodronate-containing liposomes on intestinal mucosal injury in rats with severe acute pancreatitis

BACKGROUND: Severe acute pancreatitis (SAP) can result in intestinal mucosal injury. This study aimed to demonstrate the protective effect of clodronate-containing liposomes on intestinal mucosal injury in rats with SAP. METHODS: Liposomes containing clodronate or phosphate buffered saline (PBS) were prepared by the thin-film method SAP models were prepared by a uniform injection of sodium taurocholate (2 mL/kg body weight) into the subcapsular space of the pancreas. Sprague-Dawley rats were randomly divided into a control group (C group), a SAP plus PBS-containing liposomes group (P group) and a SAP plus clodronate-containing liposomes group (T group). At 2 and 6 hours after the establishment of SAP models, 2 mL blood samples were taken from the superior mesenteric vein to measure the contents of serum TNF-α and IL-12. Pathological changes in the intestine and pancreas were observed using hematoxylin and eosin staining, while apoptosis was detected using TUNEL staining. In addition, the macrophage markers cluster of differentiation 68 (CD68) in the intestinal tissue was assessed with immunohistochemistry. RESULTS: At the two time points, the levels of TNF-α and IL-12 in the P group were higher than those in the C group (P<0.05) Compared with the P group, the levels of TNF-α and IL-12 decreased in the T group (P<0.05). The pathological scores of the intestinal mucosa and pancreas in the T group were lower than those of the P group. In the T group, large numbers of TUNEL-positive cells were observed, but none or few in the C and P groups. The number of CD68-positive macrophages decreased in the T group.CONCLUSIONS: Clodronate-containing liposomes have prote- ctive effects against intestinal mucosal injury in rats with SAP. The blockade of macrophages may provide a novel therapeutic strategy in SAP.

MRI shows clodronate-liposomes attenuating liver injury in rats with severe acute pancreatitis

BACKGROUND: Studies have revealed that macrophages play an important role in the development of severe acute pancreatitis (SAP). Activated macrophages can lead to a systemic inflammatory response, induce lipid peroxidation, impair membrane structure, result in injury to the liver and the other extrahepatic organs, and eventually result in multiple organ dysfunction syndrome by promoting excessive secretion of cytokines. Liver injury can further aggravate the systemic inflammatory response and increase mortality by affecting the metabolism of toxins and the release of excessive inflammatory mediators. Clodronate is a synthetic bisphosphonate, which is often used for treating bone changes caused by osteoporosis and other factors. In the current study, we created liposomes containing superparamagnetic iron oxide particles (SPIOs) for macrophage labeling and magnetic resonance imaging, using a novel method that can bind the clodronate to induce apoptosis and deplete macrophages. METHODS: Superparamagnetic Fe(3)O(4) nanoparticles were prepared by chemical coprecipitation. SPIO-containing liposomes and SPIO-clodronate-containing liposomes were prepared by the thin film method. SAP models were prepared by injection of sodium taurocholate (2 ml/kg body weight) into the subcapsular space of the pancreas. Sprague-Dawley rats were randomly divided into a control group, a SAP plus SPIO-liposome group, and a SAP plus SPIO-clodronate-containing group. Two and six hours after SAP models were available, T2-weighted MRI scans (in the same plane) of the livers of rats in each group were performed. At the end of the scans, 2 ml of blood was taken from the superior mesenteric vein to measure the levels of serum amylase, ALT, AST, TNF-alpha, and IL-6. Pathological changes in the liver and pancreas were assessed. RESULTS: Transmission electron microscopy showed that the liposomes had a uniform size. No pathological changes in the pancreata of rats in the control group were noted. The pathological changes in the pancreata and livers of rats in the SAP plus SPIO-clodronate-containing liposome group were milder than those in the SAP plus SPIO-liposome group. The MRI signal intensity of the livers in the SAP plus SPIO-liposome and SAP plus SPIO-clodronate-containing groups was significantly lower than that in the control group. There were significant changes in the two experimental groups (P<0.01). In addition, the levels of serum amylase, ALT, AST, TNF-alpha, and IL-6 in rats in the SAP plus SPIO-liposome group were higher than those in the control group (P<0.01), while the corresponding levels in the SPIO-clodronate-containing liposome group were significantly lower than those in the SAP plus SPIO-liposome group (P<0.01). CONCLUSION: Clodronate-containing liposomes protect against liver injury in SAP rats, and SPIO can be used as a tracer for MRI examination following liver injury in SAP rats. (Hepatobiliary Pancreat Dis Int 2010; 9: 192-200)

Clodronate-containing liposomes attenuate lung injury in rats with severe acute pancreatitis

Objectives： Severe acute pancreatitis （SAP） can lead to acute lung injury （ALl）. The purpose of this paper is to investigate the protective effect of clodronate-containing liposomes on ALl in rats with SAP. Methods： The thin film method was used to prepare liposomes. Sprague-Dawley rats were randomly divided into three groups. After the SAP model was established by injecting 5% （w/v） sodium taurocholate （2 ml/kg body weight） into the subcapsular space of the pancreata, normal saline was administered to the control （C） group, phosphate buffer solution （PBS）-containing liposome to the P group, and clodronate-containing liposome to the T group through tail veins. Blood samples were obtained from the superior mesenteric vein at 2 and 6 h to measure the levels of amylase, interleukin-6 （IL-6）, and tumor necrosis factor-a （TNF-α）. Morphological changes in the pancreata and lung were observed using hematoxylin and eosin （H＆E） staining, while cell apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL）. In addition, the macrophage marker cluster of differentiation 68 （CD68） in lung tissue was detected with immunohistochemistry. Results： Blood levels of amylase, IL-6, and TNF-α were significantly increased in the P group compared to those in the T group （P〈0.05）. In the T group, large numbers of TUNEL-positive cells were observed, but no or few in the C and P groups. Gross inspection and H＆E staining of pancreata and lung showed dramatic tissue damage, including inflammation and necrosis in the P group. Less remarkable changes were noted in the T group, and the C group exhibited normal histology. The histological scores according to Kaiser＇s criteria were consistent with H＆E findings. The number of CD68-positive macrophages decreased in the T group. Conclusions CIodronate-containing liposomes have a protective effect against ALl in rats with SAP. Blockade of macrophages may represent a novel therapeutic strategy in SAP.

Clodronate-superparamagnetic iron oxide-containing liposomes attenuate renal injury in rats with severe acute pancreatitis

Background and objective： It has been shown that macrophages play an important role in the development of severe acute pancreatitis （SAP）, and eventually lead to multiple organ failure （MOF）. Clodronate-liposome selectively depleted macrophages. This study was to investigate the role of renal macrophage infiltration in acute renal injury in rats with SAP and to evaluate the potential of superparamagnetic iron oxide （SPIO）-enhanced magnetic resonance imaging （MRI） for diagnosis. Methods： Superparamagnetic Fe3O4 nanoparticles were prepared by chemical coprecipitation. SPIO-liposomes and SPIO-clodronate-liposomes were prepared by the thin film method. SAP models were prepared by injection of sodium taurocholate into the subcapsular space of rat pancreas. Sprague-Dawley rats were randomly divided into a control group, SAP plus SPIO-liposome （P） group, and SAP plus SPIO-clodronatecontaining liposome （T） group. Kidney injury was evaluated by T2-weighted MRI scan. The levels of serum amylase （SAM）, blood urea nitrogen （BUN）, and serum creatinine （SCr） were measured by an automated enzymatic method. Serum tumor necrosis factor-α （TNF-α） was measured by enzyme-linked immunosorbent assay （ELISA）. Pathological changes in the pancreas and kidney were observed using hematoxylin and eosin （H＆E） staining, while cell apoptosis was detected with terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） staining. In addition, the macrophage markers （CD68） of the renal tissue were detected with immunohistochemistry. Results： The pathological changes in the pancreas and kidneys of rats in the T group were milder than those in the P group. The MRI signal intensity of the kidneys in the P and T groups was significantly lower than that in the control group. There were significant changes in the two experimental groups （P〈0.01）. The levels of SAM, Bun, SCr, and TNF-α in rats in the P group were higher than those in the control group （P〈0.01） and in the T group （P〈0.01）. The apoptosis of the kidney in the T group was higher than that in the P group at 2 and 6 h （P〈0.01）. Conclusions： Clodronate-containing liposomes protected against renal injury in SAP rats, and SPIO can be used as a tracer for MRI examination to detect renal injury in SAP rats. SPIO-aided MRI provided an efficient non-invasive way to monitor the migration of macrophages after renal injury in rats with SAP.